Brain specific potassium channel protein

ABSTRACT

The present invention relates to a novel potassium channel protein expressed exclusively in the brain, a DNA molecule having a sequence encoding this protein, a vector containing the DNA molecule, a host cell containing this vector, and a method for obtaining the potassium channel protein, wherein mRNA is extracted from human cells or tissue capable of producing the potassium channel protein of the present invention. Then, use is made of two primers between which the channel protein mRNA or a part of the mRNA region is located by using the thus extracted mRNA as a template. By carrying out a reverse transcriptase-polymerase chain reaction, the channel protein cDNA or a part thereof can be obtained. Thereafter, the channel protein can be produced by integrating the thus obtained novel potassium channel cDNA or a part of the same into an appropriate expression vector and then effecting its expression in a host cell. The proteins are useful (1) as targets for identifying therapeutic agents for central nervous system disorders such as dementia, cerebral ischemic disorder, epilepsy and the like; (2) for use in a method of screening compounds and peptides capable of acting on the claimed protein; and (3) as agents for the treatment of central nervous system disorders.

TECHNICAL FIELD

The present invention belongs to the field of genetic engineering, andparticularly relates to a novel potassium channel protein expressedexclusively in the brain, or an equivalent thereof, a gene encoding theprotein or an equivalent thereof, a vector containing the gene, a hostcell containing the vector, and so on.

BACKGROUND ART

Potassium channel is a protein which is distributed in the surfacemembrane of cells and selectively allows potassium ions to pass throughit, and it is considered that it takes an important role in controllingmembrane potential of cells. Particularly, in nerve and muscle cells, itcontributes to the neurotransmission of central and peripheral nerves,pace making of the heart, contraction of muscles and the like bycontrolling frequency, persistency and the like of action potential. Inaddition, it has been shown that it is also concerned in the secretionof hormones, adjustment of cell volume, proliferation of cells and thelike.

As the classification based on the opening and closing mechanism of thechannel, a voltage-dependent potassium channel, an inwardly rectifyingpotassium channel, a calcium-dependent potassium channel, a receptorcoupling type potassium channel and the like have so far beenidentified. In addition, an ATP-dependent potassium channel, apH-dependent potassium channel and the like have also been reported.Among them, the voltage-dependent potassium channel has a characteristicin that it opens when the membrane potential is depolarized. In general,potassium ions are present in a non-equilibrium state of about 5 mMoutside the cell and about 150 mM inside the cell. Thus, when thevoltage-dependent potassium channel is opened by depolarization,potassium ions flow out from intracellular part to extracellular partand, as a result, induce restoration of the membrane potential(re-polarization). Accordingly, the opening of voltage-dependentpotassium channel induces reduction of excitability of nerve and musclecells and the like. Also, it causes changes in cellular functions innon-excitatory cells too, such as increase in the driving force forCa²⁺and subsequent increase in the flow of the same ion into theintracellular part. A compound capable of modifying opening of thevoltage-dependent channel has a possibility of controlling variousfunctions of cells, including excitability of nerve and muscle cells.

Genes of some types of the voltage-dependent potassium channel have beenisolated from the brain and heart, and primary structure of the proteinhas been revealed. Based on the primary structure, it has been suggestedthat the voltage-dependent potassium channel has six transmembranedomains (S1 to S6) and one ion permeation region (H5). Also, it isassumed that the fourth transmembrane domain S4 contains basic aminoacids having positive charge at intervals of 3 to 4 bases and functionsas a voltage sensor.

These channels are roughly divided into Shaker type and eag type, basedon the similarity of amino acid sequences. The Shaker type is a familyhaving markedly high diversity and can be further divided into fourgroups of Kv1, Kv2, Kv3 and Kv4. On the other hand, the eag type isconstituted by eag, eag-related gene and elk, and it related genesinclude hyperpolarization activation type potassium channelscorresponding to KAT gene cluster and a cation channel which isactivated by a cyclic nucleotide.

Regarding the importance of voltage-dependent potassium current in thebrain, several findings have been obtained using these clonedvoltage-dependent potassium channels. For example, relationship of Kv1.1with memory and pain has been suggested by antisense-aided in vivoexperiments (Meiri, N. et al. (1997) Proc. Natl. Acad. Sci. USA, 94,4430-4434; Galeotti, N. et al. (1997) J. Pharmaco. Exp. Ther., 281,941-949). Regarding Kv3.1, its participation in the excitability of GABAactivating interneuron in cerebral cortex has been shown (Massengill, J.et al. (1997) J. Neurosci., 3136-3147). On the other hand, someexperiments carried out using tetraethylammonium and 4-aminopyridine asnon-selective inhibitors of the voltage-dependent potassium channel havealso been reported. It has been shown that tetraethylammonium suppressesvoltage-dependent potassium current in cerebral cortex nerve cells andalso inhibits apoptosis of the same nerve cells (Yu, S. P. et al.(1997), Science, 278, 114-117). Also, it is known that intraventricularadministration of 4-aminopyridine causes epileptic attack. These resultssuggest a possibility that an agent capable of controlling the activityof voltage-dependent potassium channel in the brain will become atherapeutic agent for central nervous system disorders such as dementiadue to disturbance of memory and so on, nerve cell death accompanied bycerebral ischemia, epilepsy and the like.

On the other hand, most of the voltage-dependent potassium channels sofar cloned are distributed in a large number of tissues among organs inthe whole body. Thus, even when an agent which acts selectively on aspecified voltage-dependent potassium channel is found, there is apossibility that the agent acts on many tissues and thereby inducesoriginally unexpected agent effects. In order to find an agent havingless side effects by targeting a potassium channel, it is necessary toclone a potassium channel in which its expressing tissue is restricted.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel potassiumchannel protein expressed exclusively in the brain, as a target oftherapeutic agents for central nervous system disorders such asdementia, cerebral ischemic disorder, epilepsy and the like, and anotherobject of the present invention is to provide a method for screeningcompounds and peptides capable of modifying activity of the samepotassium channel protein, which are useful as therapeutic agents forcentral nervous system disorders, and a novel agent for use in thetreatment of central nervous system disorders, which specifically actsupon the central nervous system and generates less side effects.

With the aim of solving the aforementioned problems, the presentinventors have conducted intensive studies and, as a result, succeededin isolating a gene coding for a novel potassium channel proteinexpressed exclusively in the brain. The inventors have also succeeded inexpressing the novel potassium channel protein expressed exclusively inthe brain and establishing a method for the screening of compounds andpeptides capable of modifying activity of the same potassium channelprotein.

The present invention relates to:

1) a potassium channel protein or an equivalent thereof, which has anamino acid sequence selected from either of Sequence Nos. 2 and 6, or anamino acid sequence resulting from said amino acid sequence bysubstitution, deletion or insertion of certain amino acid(s), and isexpressed exclusively in the brain,

2) the potassium channel protein or an equivalent thereof according tothe item 1), which is expressed exclusively in the human brain,

3) a potassium channel protein which has an amino acid sequence selectedfrom either of Sequence Nos. 2 and 6, 4) a gene which has a genesequence encoding the potassium channel protein or an equivalent thereofdescribed in the items 1) to 3),

5) a gene which has a gene sequence encoding an amino acid sequenceselected from either of Sequence Nos. 2 and 6, 6) a gene which has agene sequence selected from either of the 6th to 3257th gene sequence ofSequence No. 1 or the 4th to 3057th gene sequence of Sequence No. 5, ora gene which is degenerate with respect to said gene,

7) a vector which contains the gene of the items 4) to 6),

8) a host cell which contains the vector of the item 7), or

9) a method for producing the potassium channel protein described in theitems 1) to 3), which uses the host cell of the item 8).

The terms to be used in the present invention are explained in thefollowing. The term “substitution, deletion or insertion of amino acid”means that one or a plurality of amino acids are substituted, deleted orinserted in the amino acid sequence selected from either of the SequenceNos. 2 and 6.

The term “expressed exclusively in the brain” means that it is expressedin the brain but not expressed in the heart, placenta, lung, liver,skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis,ovary, small intestines, large intestine and peripheral bloodleukocytes, illustratively, it means that when Northern blotting iscarried out under the conditions of Examples, the signal is detectedonly in the brain and not detected in the heart, placenta, lung, liver,skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis,ovary, small intestines, large intestine and peripheral bloodleukocytes.

Also, the term “equivalent” means a protein having a sequence in whichone or a plurality of amino acids are substituted, deleted or insertedin the amino acid sequence of the protein which is expressed in thebrain but not expressed in the heart, placenta, lung, liver, skeletalmuscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestines, large intestine and peripheral blood leukocytes, but stillhaving the same functions when compared with the protein withoutalteration of its amino acid sequence.

The term “human origin” means that it is the same amino acid sequence ofa potassium channel protein expressed in human.

In this connection, the potassium channel and the potassium channelprotein are used as synonyms.

The novel potassium channel protein of the present invention or anequivalent thereof may be any potassium channel protein or an equivalentthereof, with the proviso that it is expressed exclusively in the brain,but is preferably a human origin. Illustratively, a potassium channelprotein or an equivalent thereof, which has an amino acid sequenceselected from either of Sequence Nos. 2 and 6, or has an amino acidsequence in which one or a plurality of amino acids in the amino acidsequence selected from either of Sequence Nos. 2 and 6 are substituted,deleted or inserted, and is expressed exclusively in the brain, isincluded in the present invention and is preferably a human origin. Morepreferred is a potassium channel protein which has an amino acidsequence selected from either of Sequence Nos. 2 and 6.

The gene which has a gene sequence encoding the novel potassium channelprotein of the present invention or an equivalent thereof may be anygene which has a gene sequence encoding a potassium channel protein oran equivalent thereof expressed exclusively in the brain, but ispreferably a gene which encodes a potassium channel protein of humanorigin. Illustratively, a gene which encodes a potassium channel proteinor an equivalent thereof having an amino acid sequence selected fromeither of Sequence Nos. 2 and 6, or a gene which encodes a potassiumchannel protein or an equivalent thereof having an amino acid sequencein which one or a plurality of amino acids in the amino acid sequenceselected from either of Sequence Nos. 2 and 6 are substituted, deletedor inserted, and is expressed exclusively in the brain, is included inthe present invention, and the gene is preferably a gene which encodes ahuman origin potassium channel protein or an equivalent thereof. Morepreferred is a gene which encodes an amino acid sequence selected fromeither of Sequence Nos. 2 and 6. Most preferred is a gene which has agene sequence selected from either of the 6th to 3257th gene sequence ofSequence No. 1 or the 4th to 3057th gene sequence of Sequence No. 5.Also included in the present invention is a gene which hybridizes withthe gene of Sequence No. 1 or 5 under a stringent condition.

Hybridization can be carried out in accordance with a known method(Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”,Cold Spring Harbor Laboratory, NY). The term “stringent condition” meansa condition that a sample after hybridization is washed twice in 2×SSCcontaining 0.1% SDS and then subjected to the following washing step.

This washing step means that the washing (65° C.) is carried out in0.5×SSC containing 0.1% SDS, preferably in 0.2×SSC containing 0.1% SDS,most preferably in 0.1×SSC containing 0.1% SDS. A gene which encodes thenovel potassium channel protein of the present invention can be obtainedby the following methods.

1) Method for Producing Novel Potassium Channel Gene

a) First Production Method

mRNA is extracted from human cells or tissue capable of producing anovel potassium channel protein. Two primers between which the channelprotein MRNA or a part of the mRNA region is located are used with thethus extracted mRNA as a template. By carrying out a reversetranscriptase-polymerase chain reaction (to be referred to as RT-PCRhereinafter), the channel protein cDNA or a part thereof can beobtained. Thereafter, the channel protein can be produced by integratingthe thus obtained novel potassium channel cDNA or a part of the sameinto an appropriate expression vector to carry out its expression in ahost cell.

Firstly, from human cells or tissue capable of producing the novelpotassium channel protein of the present invention, such as humancerebral cortex, mRNA containing that encoding the protein is extractedby a known method. Guanidine thiocyanate-hot phenol method, guanidinethiocyanate-guanidine hydrochloride method and the like can beexemplified as the extraction method, but guanidine thiocyanate-cesiumchloride method can be cited as a preferred method. The cells or tissuecapable of producing the protein can be identified, for example, by awestern blotting method which uses an antibody specific for the protein.

Purification of the MRNA can be made in accordance with a usual method,for example, the mRNA can be purified by the adsorption and elutionusing an oligo(dT)-cellulose column. The mRNA can be furtherfractionated by a sucrose density gradient centrifugation or the like.

Alternatively, a commercially available mRNA extracted sample may beused without carrying out the extraction of the mRNA.

Next, single-stranded cDNA is synthesized by carrying out reversetranscriptase reaction of the thus purified mRNA in the presence of arandom primer or an oligo dT primer. This synthesis can be carried outin the usual way. The thus obtained single-stranded cDNA is subjected toPCR using two primers between which a partial region of the gene ofinterest is located, thereby amplifying the novel potassium channel DNAof interest. The thus obtained DNA is fractionated by an agarose gelelectrophoresis or the like. As occasion demands, a DNA fragment ofinterest can be obtained by carrying out digestion of the DNA withrestriction enzymes and subsequent ligation.

b) Second Production Method

In addition to the above production method, the gene of the presentinvention can also be produced using conventional genetic engineeringtechniques. Firstly, single-stranded cDNA is synthesized using reversetranscriptase, making use of the mRNA obtained by the aforementionedmethod as a template, and then double-stranded cDNA is synthesized fromthe single-stranded cDNA. Examples of this method include SI nucleasemethod (Efstratiadis, A. et al. (1976), Cell, 7, 279-288), Land method(Land, H. et al. (1981), Nucleic Acids Res., 9, 2251-2266), O. Joon Yoomethod (Yoo, O. J. et al. (1983), Proc. Natl. Acad. Sci. USA, 79,1049-1053) and Okayama-Berg method (Okayama, H. and Berg, P. (1982),Mol. Cell. Biol., 2, 161-170).

Next, the recombinant plasmid obtained by the above method is introducedinto an Escherichia coli strain, such as DH 5α, to effect itstransformation, and then a transformant can be selected making use oftetracycline resistance or ampicillin resistance as a marker. When thehost cell is E. coli, transformation of the host cell can be carriedout, for example, by the method of Hanahan (Hanahan, D. (1983), J. Mol.Biol., 166, 557-580), namely a method in which the recombinant DNA isadded to competent cells prepared in the presence of CaCl₂ and MgCl₂ orRbCl. In this connection, phage vectors such as a lambda system or thelike can also be used as the vector in addition to a plasmid.

Regarding the method for selecting a strain containing DNA which encodesthe novel potassium channel protein of interest from the transformantsobtained above, various methods such as those shown below can beemployed.

(i) A Screening Method which uses a synthetic Oligonucleotide Probe

An oligonucleotide which corresponds to the entire portion or a part ofthe novel potassium channel protein is synthesized (in this case, it maybe either a nucleotide sequence taking the codon usage intoconsideration or a plurality of nucleotide sequences as a combination ofpossible nucleotide sequences, and in the latter case, their numbers canbe reduced by including inosine) and, using this as a probe (labeledwith ³²P or ³³P) , hybridized with transformant DNA samples denaturedand fixed on a nitrocellulose filter and the resulting positive strainsare screened and selected.

(ii) A Screening Method which uses a Probe Prepared by Polymerase ChainReaction

Oligonucleotides of sense primer and antisense primer corresponding to apart of the novel potassium channel protein are synthesized, and a DNAfragment which encodes the entire portion or a part of the novelpotassium channel protein of interest is amplified by carrying outpolymerase chain reaction (Saiki, R. K. et al. (1988), Science, 239,487-491) using these primers in combination. As the template DNA to beused herein, cDNA synthesized by reverse transcription reaction frommRNA of cells capable of producing the novel potassium channel protein,or genomic DNA, can be used. The thus prepared DNA fragment is labeledwith ³²P or ³³P, and the clone of interest is selected by carrying outcolony hybridization or plaque hybridization using this fragment as aprobe.

(iii) A Method in which Screening is Carried out by Producing the NovelPotassium Channel Protein in Other Animal Cells

Genes are amplified by culturing the transformants, and transfection ofanimal cells with the genes is carried out (in this case, either aplasmid which can replicate by itself and contains a transcriptionpromoter region or a plasmid which can be integrated into the chromosomeof animal cells may be used), thereby effecting production of proteinsencoded by the genes on the cell surface. A strain containing cDNA whichencodes the novel potassium channel protein of interest is selected bydetecting the protein using an antibody for the novel potassium channelprotein, or from the original transformants using the channel activityas a marker.

(iv) A Method in which the Selection is Carried out Using ChannelActivity for the Novel Potassium Channel Protein as a Marker

cDNA is integrated into an expression vector in advance, proteins areproduced on the cell surface of transformants, and the strain ofinterest is selected by detecting desired novel potassium channelprotein producing strains using the channel activity as a marker.

(v) A Method in which the Selection is Carried out Using an Antibody forthe Novel Potassium Channel Protein

cDNA is integrated into an expression vector in advance, proteins areproduced on the cell surface of transformants, and the strain ofinterest is selected by detecting desired novel potassium channelprotein producing strains using an antibody for the novel potassiumchannel protein and a second antibody for the first antibody.

(vi) A Method which uses a Selective Hybridization Translation System

cDNA obtained from each transformant is blotted for example on anitrocellulose filter and hybridized with mRNA prepared from the novelpotassium channel protein producing cells, and then the mRNA bonded tothe cDNA is dissociated and recovered. The thus recovered MRNA istranslated into protein in a protein translation system such asinjection into Xenopus oocyte or a cell-free system such as a rabbitreticulocyte lysate, wheat germ or the like. A strain of interest isselected by the detection using an antibody for the novel potassiumchannel protein.

The method for collecting DNA which encodes the novel potassium channelprotein from the thus obtained transformant of interest can be carriedout in accordance with a known method (Maniatis, T. et al. (1982):“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY). For example, it can be made by separating a fraction correspondingto the plasmid DNA from cells and cutting out the cDNA region from theplasmid DNA.

The method for collecting DNA which encodes the novel potassium channelprotein from the thus obtained transformant of interest can be carriedout in accordance with a known method (Maniatis, T. et al. (1982):“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY). For example, it can be made by separating a fraction correspondingto the plasmid DNA from cells and cutting out the cDNA region from theplasmid DNA.

c) Third Production Method

The DNA having a nucleotide sequence which encodes the amino acidsequence selected from either of Sequence Nos. 2 and 6 can also beproduced by binding a gene fragment produced by a chemical synthesismethod. Each DNA can be synthesized using a DNA synthesizer (e.g., Oligo1000M DNA Synthesizer (Beckman) or 394 DNA/RNA Synthesizer (AppliedBiosystems)).

d) Fourth Production Method

In order to effect expression of the novel potassium channel proteinfunction expressed exclusively in the brain by the substance obtained bygenetic engineering techniques making use of the DNA of the presentinvention, it is not always necessary to have entire portion of theamino acid sequence selected from either of Sequence Nos. 2 and 6; forexample, when a part of the sequence shows functions of the novelpotassium channel protein expressed exclusively in the brain, such anamino acid sequence is also included in the potassium channel protein ofthe present invention. Also, as is known in the interferon gene and thelike, it is considered that genes of eucaryote generally showpolymorphism (e.g., see Nishi, T. et al. (1985), J. Biochem., 97,153-159), so that there will be a case in which one or a plurality ofamino acids are substituted due to the polymorphism or a case in whichthe nucleotide sequence is changed but the amino acids are not changed.In consequence, even in the case of a protein in which one or aplurality of amino acid residues are substituted, deleted or inserted atone or a plural positions in the amino acid sequence selected fromeither of Sequence Nos. 2 and 6, it is possible that it has the channelactivity and is expressed exclusively in the brain. In the presentinvention, such a protein is called an equivalent of the novel potassiumchannel protein.

All genes which encode such equivalents of the novel potassium channelprotein and have equivalent nucleotide sequences are included in thepresent invention. These various types of DNA of the present inventioncan also be produced by nucleic acid chemical synthesis in accordancewith a usual method such as phosphite triester method (Hunkapiller, M.et al. (1984), Nature, 10, 105-111), based on the information on theaforementioned novel potassium channel protein. In this connection,codons for each amino acid are well known and can be optionally selectedand determined in the usual way, for example by taking codon usage ofeach host to be used into consideration (Crantham, R. et al. (1981),Nucleic Acids Res., 9, r43-r74). In addition, partial modification ofcodons of these nucleotide sequences can be carried out in accordancewith a usual method such as site specific mutagenesis which uses aprimer comprised of a synthetic oligonucleotide coding for a desiredmodification (Mark, D. F. et al. (1984), Proc. Natl. Acad. Sci. USA, 81,5662-5666).

Determination of the DNA sequences obtained by the above methods a) tod) can be carried out by, for example, the Maxam-Gilbert chemicalmodification method (Maxam, A. M. and Gilbert, W. (1980): “Methods inEnzymology” 65, 499-559) or the dideoxynucleotide chain terminationmethod which uses M13 (Messing, J. and Vieira, J (1982), Gene, 19,269-276).

Also, the vector of the present invention, the host cell of the presentinvention and the potassium channel protein of the present invention canbe obtained by the following methods.

2) Method for Producing Recombinant Protein of the Novel PotassiumChannel

An isolated fragment containing a gene coding for the novel potassiumchannel protein can transform a host cell of other eucaryote byre-integrating it into an appropriate vector DNA. Also, it is possibleto effect expression of the gene in respective host cell, by introducingan appropriate promoter and a sequence related to the gene expressioninto the vector.

Cells of vertebrates, insects, yeast and the like are included in theeucaryotic host cells, and COS cell as a simian cell (Gluzman, Y.(1981), Cell, 23, 175-182), a dihydrofolate reductase defective strainof Chinese hamster ovary cell (CHO) (Urlaub, G. and Chasin, L.A. (1980),Proc. Natl. Acad. Sci. USA, 77, 4216-4220) and the like are frequentlyused as the vertebral cells, though not limited thereto.

The expression vector which can be used in vertebral cells generally hasa promoter positioned at the upstream of the gene to be expressed, anRNA splicing region, a polyadenylation region, a transcriptiontermination sequence and the like, and it may further have a replicationorigin as occasion demands. Examples of the expression vector includepSV2dhfr which has SV40 early promoter (Subramani, S. et al. (1981),Mol. Cell. Biol., 1, 854-864), though not limited thereto.

When COS cell is used as the host cell, a vector which has the SV40replication origin, can perform autonomous replication and has atranscription promoter, a transcription termination signal and an RNAsplicing region can be used as the expression vector, and its examplesinclude pME18S (Maruyama, K. and Takebe, Y. (1990), Med. Immunol., 20,27-32), pEF-BOS (Mizushima, S. and Nagata, S. (1990), Nucleic AcidsRes., 18, 5322), pCDM8 (Seed, B. (1987), Nature, 329, 840-842) and thelike. The expression vector can be incorporated into COS cell, forexample, by DEAE-dextran method (Luthman, H. and Magnusson, G. (1983),Nucleic Acids Res., 11, 1295-1308), calcium phosphate-DNAco-precipitation method (Graham, F. L. and van der Ed, A. J. (1973),Virology, 52, 456-457) or electroporation (Neumann, E. et al. (1982),EMBO J., 1, 841-845), thereby obtaining a desired transformant cell.Also, when CHO cell is used as the host cell, a transformant cellcapable of stably producing the novel potassium channel protein can beobtained by carrying out co-transfection of a expression vector togetherwith a vector capable of expressing neo gene which functions as a G418resistance marker, such as pRSVneo (Sambrook, J. et al. (1989):“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY) or pSV2-neo (Southern, P. J. and Berg, P. (1982), J. Mol. Appl.Genet., 1, 327-341) or the like, and selecting G418 resistant colonies.

The thus obtained transformant of interest can be cultured in the usualway, and the novel potassium channel protein is produced inside thecells or on the cell surface. Regarding the medium to be used in theculturing, it can be optionally selected from various commonly usedtypes depending on the host cell used, and in the case of theaforementioned COS cell for example, a medium such as RPMI-1640 medium,Dulbecco's modified Eagle's minimum essential medium (DMEM) or the likecan be used by supplementing it with serum component such as fetalbovine serum (FBS) or the like as occasion demands.

The novel potassium channel protein thus produced in the cells or on thecell surface of the transformant can be separated and purified therefromby various known separation techniques making use of the physicalproperties, chemical properties and the like of the channel protein.Illustrative examples of such techniques, to be carried out aftersolubilization of the channel protein-containing membrane fraction,include usual treatment with a protein precipitant, ultrafiltration,various liquid chromatography techniques such as molecular sievechromatography (gel filtration), adsorption chromatography, ion exchangechromatography, affinity chromatography, high performance liquidchromatography (HPLC) and the like, dialysis and combinations thereof.In this connection, the membrane fraction can be obtained in the usualway. For example, it can be obtained by culturing the cells whichexpressed the novel potassium channel protein on the cell surface,suspending them in a buffer and then homogenizing and centrifuging them.Also, when the channel protein is solubilized using a solubilizing agentas mild as possible (CHAPS, Triton X-100, digitonin or the like),characteristics of the channel can be maintained after thesolubilization.

A method for the screening of compounds and peptides capable ofmodifying activities of the potassium channel protein is included in thepresent invention. This screening method includes a means of adding anagent to be tested to the system and measuring the index, in which anindex of the modification of potassium channel protein in response to aphysiological characteristic of the potassium channel protein ismeasured making use of the thus constructed potassium channel proteinwhich is expressed exclusively in the brain. The following screeningmethods can be cited as illustrative examples of this measuring system.Also, examples of the agents to be tested include compounds or peptideswhich are conventionally known to have potassium channel ligandactivities but their ability to selectively modify activities of thepotassium channel protein expressed exclusively in the brain is notclear (e.g., compounds described in JP-A-4-178375; the term “JP-A” asused herein means an “unexamined published Japanese patentapplication”), various known compounds and peptides registered inchemical files but their potassium channel ligand activities areunknown, compounds obtained by combinatorial chemistry techniques andrandom peptides prepared by employing a method such as phage display andthe like. In addition, culture supernatants of microorganisms andnatural components originated from plants and marine organisms are alsoobjects of the screening. Also useful are compounds or peptides obtainedby chemically or biologically modifying a compound or peptide selectedby the screening method of the present invention.

3) Method for the Screening of Compounds and Peptides Capable ofModifying Activities of the Novel Potassium Channel Protein

a) Screening Method to which Voltage-Clamp Method is Applied

Channel activities of the novel potassium channel protein can bemeasured by the whole-cell voltage-clamp method. Cells in which thechannel protein is expressed are subjected to membrane potentialfixation by the whole-cell voltage-clamp method and whole-cell currentis measured. A solution containing 145 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂and 0.8 mM MgCl₂ is used as the extracellular liquid, and a solutioncontaining 155 mM KCl is used as the intracellular liquid (patchelectrode solution). Compounds and peptides capable of modifyingactivities of the novel potassium channel protein can be screened bycomparing outward currents generated by a depolarization stimulus,namely shifting of membrane potential from the holding potential (e.g.,−70 mV) to the depolarization side (e.g., −80 mV), in the presence andabsence of an agent to be tested.

b) Screening Method utilizing Rb⁺ion Release

In general, the potassium channel can pass Rb⁺ion similar to K⁺ion, sothat its channel activity can be measured using radioactive isotope⁸⁶Rb⁺release as an index. By carrying out incubation of novel potassiumchannel protein-expressed cells together with ⁸⁶RbCl (e.g., 18 hr at 37°C.), ⁸⁶Rb⁺can be incorporated into the cells. The cells are washed withlow K⁺concentration physiological saline (e.g., 4.5 mM K⁺) and thensuspended in the same solution. When a high K⁺concentration solution(e.g., 100 mM in final concentration) is added to the cell suspension,membrane potential of the cells is depolarized and the potassium channelis activated. Since ⁸⁶Rb⁺inside the cells is thereby released into theextracellular moiety, the radioactivity in the extracellular liquid canbe used as an index of the channel activity. Compounds and peptidescapable of modifying activity of the novel potassium channel protein canbe screened by comparing radioactivity released into the extracellularmoiety when the high K⁺concentration solution is added, in the presenceand absence of an agent to be tested.

c) Screening Method which uses Voltage-Sensitive Ddye or IntracellularK⁺Detecting Dye

A voltage-sensitive dye or a intracellular K⁺detecting dye can opticallydetect changes in the membrane potential or intracellularK⁺concentration accompanied by the opening of the potassium channel. Asthe voltage-sensitive dye, RH155, WW781, Di-4-ANEPPS or derivativesthereof can be used. Also, a chimeric protein prepared by inserting theamino acid sequence of green fluorescent protein into the C-terminalintracellular region of Shaker type voltage-dependent potassium channelcan be used for the detection of membrane potential (Siegel, M. S. andIsacoff, E. Y. (1997), Neuron, 19, 735-741). As the intracellularK⁺detecting dye, K⁺-binding benzofuran isophthalate and the like can beused. Since channel activity of the novel potassium channel can bemeasured by the use of these dyes, compounds and peptides capable ofmodifying activity of the novel potassium channel protein can bescreened by comparing the changing amounts in the presence and absenceof an agent to be tested.

A medicament which contains a compound or peptide capable ofsignificantly modifying the activity of potassium channel proteinexclusively expressed in the brain, selected by the aforementionedscreening as an active ingredient, is included in the present invention.

The medicament of the present invention is characterized in that it hasnovel pharmacological action to selectively control activities ofpotassium channel in the brain, and the use of the medicament is fordiseases caused by abnormalities (e.g., acceleration, reduction, anddenaturation) of potassium channel activities in the brain or thosewhich express and complicate such abnormalities, wherein theirillustrative examples include central nervous system disorders such asdementia, cerebral ischemic disorder, epilepsy and the like.

The pharmaceutical preparation which contains a compound or peptidecapable of modifying activity of the potassium channel protein of thepresent invention, as an active ingredient, can be prepared usingcarriers, fillers and other additives generally used in the preparationof medicaments, in response to each type of the active ingredient.

Examples of its administration include oral administration by tablets,pills, capsules, granules, fine granules, powders, oral solutions andthe like, and parenteral administration by injections (e.g.,intravenous, intramuscular or the like), suppositories, transdermalpreparations, transmucosal absorption preparations and the like.Particularly, in the case of peptides which are digested by gastricacid, parenteral administration such as intravenous injection or thelike, or lower gastrointestinal delivery administration is desirable.

In the solid composition for use in the oral administration according tothe present invention, one or more active substances are mixed with atleast one inert diluent such as lactose, mannitol, glucose,microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone or aluminum magnesium silicate. In the usual way, thecomposition may contain other additives than the inert diluent, such asa lubricant, a disintegrating agent, a stabilizing agent and asolubilizing or solubilization assisting agent. If necessary, tablets orpills may be coated with a sugar coating or a film of a gastric orenteric substance.

The liquid composition for oral administration includes emulsions,solutions, suspensions, syrups and elixirs and contains a generally usedinert diluent such as purified water or ethyl alcohol. In addition tothe inert diluent, this composition may also contain other additivessuch as moistening agents, suspending agents, sweeteners, flavors andantiseptics.

The injections for parenteral administration includes aseptic aqueous ornon-aqueous solutions, suspensions and emulsions. Examples of thediluent for use in the aqueous solutions and suspensions includedistilled water for injection use and physiological saline. Examples ofthe diluent for use in the non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, plant oil (e.g., olive oil or thelike), alcohols (e.g., ethanol or the like), polysorbate 80 and thelike. Such a composition may further contain a moistening agent, anemulsifying agent, a dispersing agent, a stabilizing agent, asolubilizing or solubilization assisting agent, an antiseptic and thelike. These compositions are sterilized for example by filtrationthrough a bacteria retaining filter, blending of a germicide orirradiation. Alternatively, they may be used by firstly making intosterile solid compositions and dissolving them in sterile water or othersterile solvent for injection use prior to their use.

The dose is optionally decided by taking into consideration strength ofeach active ingredient selected by the aforementioned screening methodand symptoms, age, sex and the like of each patient to be administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of Sequence No. 2 (upper line) andSequence No. 6 (lower line) potassium channels.

FIG. 2 shows results of Northern analysis on the novel potassium channelin each human organ. A and B represent results on the potassium channelof Sequence No. 2, and C and D on the potassium channel of Sequence No.6.

FIG. 3 shows results of Northern analysis on the novel potassium channelin each region of the human brain. A and B represent results on thepotassium channel of Sequence No. 2, and C and D on the potassiumchannel of Sequence No. 6.

In FIG. 4, A and B show results of in situ hybridization regardingpotassium channel (Sequence No. 2) (DG, the granule cell layer of thedentate gyrus; CA1 and CA3, the pyramidal cell layer of the CA1 and CA3fields of Ammon's horn; Cx, cerebral cortex). C and D represent enlargedimages of cerebral cortex (arrows indicate signals in typical pyramidalcells). An antisense probe (in A and C) or a sense probe (in B and D)was used in the hybridization. The scale bar indicates 1.5 mm (A and B)or 50 μm (B and D).

FIG. 5 shows results of the detection of the channel activity ofpotassium channel (Sequence No. 2) by depolarization stimulus.

FIG. 6 shows results of the detection of the channel activity of potassium channel (Sequence No. 6) by depolarization stimulus.

FIG. 7 shows tail current of the potassium channel shown in Sequence No.2.

FIG. 8 shows current response of the pot assium channel shown inSequence No. 6 in high potassium extracellular liquid.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to disclose the present invention more illustratively, examplesare described in the following, but the present invention is not limitedto the examples. In this connection, unless otherwise noted, they can becarried out in accordance with known methods (Maniatis, T. et al.(1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring HarborLaboratory, NY).

EXAMPLE 1 Isolation of Gene Encoding the Novel Potassium Channel Protein

The full-length CDNA encoding the novel potassium channel protein wasobtained by RT-PCR using human brain poly A⁺RNA (Clontech) as atemplate.

In order to isolate gene of the potassium channel protein shown inSequence No. 2, 5′-GGAATTCC CTA AGA TGC CGG CCA TGC-3′ (Sequence No. 3)was used as a forward primer, and 5′-GCTCTAGAGC ACT CTG AGG TTG GGC CGAAC-3′ (Sequence No. 4) as a reverse primer (EcoRI site and XbaI site areadded to respective 5′-termini). RT-PCR was carried out by Hot Startmethod using Pfu DNA polymerase (Stratagene). After carrying out firstthermal denaturation at 96° C. (1 minute), a cycle of 96° C. (10seconds)/68° C. (30 seconds)/72° C. (7 minutes) was repeated 35 times.As a result, a DNA fragment of about 3.3 kbp was amplified. Thisfragment was digested with EcoRI and xbaI and then cloned using pME18Splasmid. Since the pME18S plasmid contains SR promoter which showsstrong promoter activity in animal cells (Takebe, Y. et al. (1988), Mol.Cell. Biol., 8, 466-472), it can be used for expressing recombinantprotein in animal cells. This plasmid was received from Dr. Saito atChiba University (Maruyama, K and Takebe, Y. (1990), Med. Immunol., 20,27-32). Nucleotide sequence of the thus obtained clone pME-E1 wasanalyzed by dideoxy terminator method using ABI377 DNA Sequencer(Applied Biosystems). The thus revealed sequence is shown in SequenceNo. 1 of Sequence Listing.

This sequence has a 3252-base open reading frame (from the 6th to 3257thof Sequence No. 1). The amino acid sequence (1083 amino acids) deducedfrom the open leading frame is shown in Sequence No. 2 in the SequenceListing.

In order to isolate gene of the potassium channel protein shown inSequence No. 6, 5′-GCC ATG CCG GTC ATG AAG G-3′ (Sequence No. 7) wasused as a forward primer, and 5′-GCC AGG GTC AGT GGA ATG TG-3′ (SequenceNo. 8) as a reverse primer. RT-PCR was carried out by Hot Start methodusing TaKaRa LA Taq (Takara Shuzo). After carrying out first thermaldenaturation at 98° C. (1 minute), a cycle of 98° C. (15 seconds)/68° C.(3 minutes) was repeated 35 times and then finally 10 minutes ofextension reaction was carried out at 72° C. As a result, a DNA fragmentof about 3.1 kbp was amplified. This fragment was cloned using pCR2.1plasmid (Invitrogen). Nucleotide sequence of the thus obtained clone isshown in Sequence No. 5 of Sequence Listing. In order to express thegene in animal cells, it was subcloned in pME18S plasmid and namedpME-E2. The Sequence No. 5 contains an open reading frame of 3,054 bases(from 4th to 3057th positions of Sequence No. 5). Amino acid sequencededuced from the open reading frame (1,017 amino acids) is shown inSequence No. 6 of Sequence Listing.

Both of the amino acid sequences of the potassium channels have sixhydrophobic regions considered to be transmembrane domains (S1 to S6)which are characteristic of voltage-dependent potassium channel. Also,the S4 domain which is considered to be a voltage sensor has acharacteristic in that basic amino acids are continued at an interval of3 amino acids, and a moderately basic sequence which corresponds to theH5 region is also present between S5 and S6. In addition, both aminoacid sequences have high mutual homology. Results of the alignment ofboth amino acid sequences are shown in FIG. 1. Their homology was 48%with the entire amino acid sequences and 70% with the hydrophobicregions (from 227 position Trp to 508 position Tyr of Sequence No. 2 andfrom 229 position Trp to 482 position Tyr of Sequence No. 6). In thiscase, MegAlign program of an analyzing software Lasergene (DNASTAR) wasused in the analyses of sequence alignment and homology.

EXAMPLE 2 Distribution of Novel Potassium Channel Gene Expression inHuman Tissues

Distribution of the novel potassium channel gene expression was analyzedby Northern blot hybridization. A cDNA fragment corresponding to theC-terminal intracellular region (from 2105th to 2956th positions ofSequence No. 1 for the potassium channel of Sequence No. 2, or from2241st to 2898th positions of Sequence No. 5 for the potassium channelof Sequence No. 6) was used as a probe. Hybridization of the probe witha membrane on which poly A⁺RNA (2 μg) of each human organ had beenblotted was carried out at 42° C. (18 hours) in a solution containing50% formamide, 5×SSPE, 10×Denhardt's solution, 2% SDS and 100 μg/ml ofdenatured salmon sperm DNA. The membrane was finally washed twice with asolution containing 0.1×SSC and 0.1% SDS (at 55° C. on the potassiumchannel of Sequence No. 2 and at 60° C. on the potassium channel ofSequence No. 6, each for 30 minutes).

When Northern analysis was carried out on various human organs (theheart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,spleen, thymus, prostate, testis, ovary, small intestines, largeintestine and peripheral blood leukocyte), a signal of about 4 kb on thepotassium channel of Sequence No. 2 and signals of about 4.4 kb andabout 7.5 kb on the potassium channel of Sequence No. 6 were detected,all in the brain alone (FIG. 2). That is, it was found that the mRNA ofboth of the novel potassium channels is expressed exclusively in thebrain. In this connection, selective expression of the Sequence No. 6potassium channel mRNA in the brain was confirmed also by RT-PCRanalysis.

In addition, the Northern analysis was carried out also on variousregions of the human brain (cerebellum, cerebral cortex, medulla, spinalcord, cerebral cortex occipital lobe, cerebral cortex frontal lobe,cerebral cortex temporal lobe, putamen, amygdala, caudate nucleus,corpus callosum, hippocampus, substantia nigra, subthalamic nucleus andthalamus). It was found that MRNA of the potassium channel shown inSequence No. 2 is expressed exclusively in the telencephalon includingcerebral cortex, caudate nucleus, hippocampus and striatum (putamen andcaudate nucleus) (FIG. 3 A and B). On the other hand, it was found thatmRNA of the potassium channel shown in Sequence No. 6 is expressedfrequently in striped body and cerebral cortex (FIG. 3 C and D). Also,weak expression was found in hippocampus and amygdala. Distributions ofboth potassium channel genes were overlapped.

EXAMPLE 3 Distribution of Novel Potassium Channel Expression in Neuronsof the Central Nervous System

In order to examine expression of the novel potassium channel gene inneurons of the central nervous system, in situ hybridization analysis ofrat brain sections was carried out. The in situ hybridization wascarried out in accordance with a report (Okumura, K. et al. (1997),Oncogene, 14, 713-720) using an antisense RNA probe labeled withdigoxigenin. A sense probe was used in the control test. These probeswere prepared based on a rat potassium channel gene sequence revealed bythe following procedure.

In order to obtain partial sequences of rat potassium channel genes,RT-PCR was carried out using 5′-ACC TTC CTG GAC ACC ATC GC-3′ (SequenceNo. 11) and 5′-CCA AAC ACC ACC GCG TGC AT-3′ (Sequence No. 12) asprimers. Both primers correspond to a region of from 14 to 20 positionsand a region of from 493 to 499 positions of the amino acid sequence ofSequence No. 2, respectively. As a result of carrying out RT-PCR usingpoly A⁺RNA isolated from rat brain as a template, fragments of about 1.5kb and about 1.4 kb respectively corresponding to rat orthologous genesof the potassium channels described in Sequence No. 2 and Sequence No. 6were obtained. Next, based on the nucleotide sequences revealed from therespective fragments, RACE was carried out on them to reveal completelength sequences. RACE was carried out using Rat Brain Marathon-ReadycDNA (Clontech) in accordance with the manufacturer's instruction.Sequence of the rat orthologous gene of the potassium channel describedin Sequence No. 2 is shown in Sequence No. 9, and sequence of the ratorthologous gene of the potassium channel described in Sequence No. 6 isshown in Sequence No. 10.

An antisense sequence or sense sequence of from 2,683 to 3,204 positionsof Sequence No. 9 was used as a probe for the aforementioned ratorthologous gene of the potassium channel described in Sequence No. 2.As a result of the in situ hybridization, signals were observed inhippocampus only when the antisense probe was used, and the expressionwas confirmed in the same region (FIG. 4 A and B). The expression inhippocampus was observed in granule cells of the dentate gyrus,pyramidal cells of the CA1 and CA3 fields of Ammon's horn and the likeneurons. Specific signals were also observed in pyramidal cells asneurons of cerebral cortex (FIG. 4 C and D). Thus, it was confirmed thatthe potassium channel is expressed in the neurons of the central nervoussystem.

A sequence of from 3,140 to 3,750 positions of Sequence No. 10 was usedas a probe for the aforementioned rat orthologous gene of the potassiumchannel described in Sequence No. 6. It was found that this potassiumchannel was strongly expressed in neurons of the cerebral cortex.

EXAMPLE 4 Induction of the Expression of Novel Potassium Channel Protein

In order to detect channel activity of the novel potassium channelprotein, expression of the protein was induced in an animal cell. As thecell, L929 cell which does not generate current by intrinsic channelthrough changes in the membrane potential was used. Transfection of thecell was carried out by lipofectAMINE method using pME-E1 plasmid orpME-E2 plasmid.

EXAMPLE 5 Detection of Channel Activity of Novel Potassium ChannelProtein

The transfected cell was voltage-clamped and whole-cell current wasmeasured using the whole-cell voltage-clamp method. A solutioncontaining 140 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 0.8 mM MgCl₂, 15 mMglucose and 10 mM HEPES-Na (pH=7.4) was used as the extracellularsolution, and a solution containing 125 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂,11 mM EGTA and 10 mM HEPES-K (pH=7.2) was used as the intracellularsolution.

The cell transfected with pME-E1 was depolarized to voltages between −40mV and +80 mV at 20 mV intervals for 200 msec from the holding potentialof −70 mV (FIG. 5), or the cells transfected with pME-E2 was depolarizedto voltages between −60 mV and +60 mV at 20 mV intervals for 200 msecfrom the holding potential of −120 mV (FIG. 6). As a result, distinctiveoutward current was induced in both cells. It was found from this resultthat both of the protein shown in Sequence No. 2 and the protein shownin Sequence No. 6 are voltage-dependent channels.

EXAMPLE 6 Selectivity of K⁺ion by Novel Potassium Channel Protein

In order to examine selectivity of K⁺ion by the potassium channel shownin Sequence No. 2, tail current was measured. The current was examinedby a method similar to Example 5. Based on the tail current (activationof the channel by depolarization stimulus of +80 mV for 200 msec fromthe holding potential of −70 mV and then re-polarization to voltagesbetween −120 mV and −20 mV at 20 mV intervals), the reversal potentialin this solution was found to be −80 mV (FIG. 7). Since this valuealmost coincided with the equilibrium potential of K⁺obtained by theformula of Nernst (−87 mV, 25° C.; [K]out=5.4 mM, [K]in=158 mM), thischannel was considered to have large selectivity for K⁺ion.

An extracellular solution containing 155 mM KCl, 4.5 mMN-methyl-D-glucamine, 2 mM CaCl₂, 10 mM glucose and 10 mM HEPES (pH=7.4)was used for the examination of K⁺selectivity of the potassium channelshown in Sequence No. 6. Other conditions were examined by a methodsimilar to Example 5. When depolarization stimulus (between −20 mV and+20 mV at 10 mV intervals for 200 msec from the holding potential of−120 mV) was carried out, the current response was reversed at 0 mV(FIG. 8). It was estimated from this result that the reverse potentialwas approximately 0 mV. Since this value almost coincided with theequilibrium potential of K⁺obtained by the formula of Nernst (−5 mV, 25°C.; [K]out=155 mM, [K]in=158 mM), and outward current was induced inExample 5, this channel was considered to have large selectivity forK⁺ion.

Industrial Applicability

A novel potassium channel protein expressed exclusively in the brain, agene encoding this protein, a vector containing this gene, a host cellcontaining this vector and a method for producing this potassium channelprotein were provided by the present invention.

Also, there was provided a method for the screening of new medicaments,particularly new therapeutic agents for central nervous systemdisorders, in which compounds and peptides capable of modifying activityof the potassium channel protein are screened by allowing the potassiumchannel protein of the present invention to contact with agents to betested.

For example, among tissues in which the potassium channel of the presentinvention shown in Sequence No. 2 was expressed as the result of Example3, hippocampus is a region where its relationship with memory andlearning is strongly suggested (Lavitan, I. B. and Kaczmarek, L. K.(1991), The Neuron: Cell and Molecular Biology, Oxford University Press,New York, N.Y.). Particularly, granule cells of the dentate gyrus andpyramidal cells of the CA1 and CA3 fields in which expression of thepotassium channel was confirmed form a neural circuit, and variousmemory inputs are transmitted from the granule cells of the dentategyrus to pyramidal cells of the CA3 field via pyramidal cells of the CA1field, mediated by an excitatory synapse which uses glutamic acid as aneurotransmitter. It is considered that long-term changes in theefficiency of synaptic transmission observed in respective synapse, suchas long-term increase and long-term repression, are deeply concerned inmemory and learning. These long-term changes are controlled by theexcitation frequency and excitation strength of nerve cells, andvoltage-dependent potassium channels generally have a possibility ofbeing able to control excitability of neurons, so that the potassiumchannel protein of the present invention which is a voltage-dependentpotassium channel has a possibility that it is concerned in theformation of memory and learning via the excitability control ofneurons.

Regarding the medicament which contains a compound or peptide capable ofspecifically modifying activity of the potassium channel protein of thepresent invention as its active ingredient, its usefulness is expectedfor example as a therapeutic agent for central nervous system disorderssuch as dementia, cerebral ischemic disorders and epilepsy, which actscentral nervous system-specifically and has less side effects.

12 1 3323 DNA Homo sapiens CDS (6)..(3257) 1 ctaag atg ccg gcc atg cggggc ctc ctg gcg cct cag aac acc ttc ctg 50 Met Pro Ala Met Arg Gly LeuLeu Ala Pro Gln Asn Thr Phe Leu 1 5 10 15 gac acc atc gct acg cgc ttcgac ggc acg cac agt aac ttc gtg ctg 98 Asp Thr Ile Ala Thr Arg Phe AspGly Thr His Ser Asn Phe Val Leu 20 25 30 ggc aac gcc cag gtg gcg ggg ctcttc ccc gtg gtc tac tgc tct gat 146 Gly Asn Ala Gln Val Ala Gly Leu PhePro Val Val Tyr Cys Ser Asp 35 40 45 ggc ttc tgt gac ctc acg ggc ttc tcccgg gct gag gtc atg cag cgg 194 Gly Phe Cys Asp Leu Thr Gly Phe Ser ArgAla Glu Val Met Gln Arg 50 55 60 ggc tgt gcc tgc tcc ttc ctt tat ggg ccagac acc agt gag ctc gtc 242 Gly Cys Ala Cys Ser Phe Leu Tyr Gly Pro AspThr Ser Glu Leu Val 65 70 75 cgc caa cag atc cgc aag gcc ctg gac gag cacaag gag ttc aag gct 290 Arg Gln Gln Ile Arg Lys Ala Leu Asp Glu His LysGlu Phe Lys Ala 80 85 90 95 gag ctg atc ctg tac cgg aag agc ggg ctc ccgttc tgg tgt ctc ctg 338 Glu Leu Ile Leu Tyr Arg Lys Ser Gly Leu Pro PheTrp Cys Leu Leu 100 105 110 gat gtg ata ccc ata aag aat gag aaa ggg gaggtg gct ctc ttc cta 386 Asp Val Ile Pro Ile Lys Asn Glu Lys Gly Glu ValAla Leu Phe Leu 115 120 125 gtc tct cac aag gac atc agc gaa acc aag aaccga ggg ggc ccc gac 434 Val Ser His Lys Asp Ile Ser Glu Thr Lys Asn ArgGly Gly Pro Asp 130 135 140 aga tgg aag gag aca ggt ggt ggc cgg cgc cgatat ggc cgg gca cga 482 Arg Trp Lys Glu Thr Gly Gly Gly Arg Arg Arg TyrGly Arg Ala Arg 145 150 155 tcc aaa ggc ttc aat gcc aac cgg cgg cgg agccgg gcc gtg ctc tac 530 Ser Lys Gly Phe Asn Ala Asn Arg Arg Arg Ser ArgAla Val Leu Tyr 160 165 170 175 cac ctg tcc ggg cac ctg cag aag cag cccaag ggc aag cac aag ctc 578 His Leu Ser Gly His Leu Gln Lys Gln Pro LysGly Lys His Lys Leu 180 185 190 aat aag ggg gtg ttt ggg gag aaa cca aacttg cct gag tac aaa gta 626 Asn Lys Gly Val Phe Gly Glu Lys Pro Asn LeuPro Glu Tyr Lys Val 195 200 205 gcc gcc atc cgg aag tcg ccc ttc atc ctgttg cac tgt ggg gca ctg 674 Ala Ala Ile Arg Lys Ser Pro Phe Ile Leu LeuHis Cys Gly Ala Leu 210 215 220 aga gcc acc tgg gat ggc ttc atc ctg ctcgcc aca ctc tat gtg gct 722 Arg Ala Thr Trp Asp Gly Phe Ile Leu Leu AlaThr Leu Tyr Val Ala 225 230 235 gtc act gtg ccc tac agc gtg tgt gtg agcaca gca cgg gag ccc agt 770 Val Thr Val Pro Tyr Ser Val Cys Val Ser ThrAla Arg Glu Pro Ser 240 245 250 255 gcc gcc cgc ggc ccg ccc agc gtc tgtgac ctg gcc gtg gag gtc ctc 818 Ala Ala Arg Gly Pro Pro Ser Val Cys AspLeu Ala Val Glu Val Leu 260 265 270 ttc atc ctt gac att gtg ctg aat ttccgt acc aca ttc gtg tcc aag 866 Phe Ile Leu Asp Ile Val Leu Asn Phe ArgThr Thr Phe Val Ser Lys 275 280 285 tcg ggc cag gtg gtg ttt gcc cca aagtcc att tgc ctc cac tac gtc 914 Ser Gly Gln Val Val Phe Ala Pro Lys SerIle Cys Leu His Tyr Val 290 295 300 acc acc tgg ttc ctg ctg gat gtc atcgca gcg ctg ccc ttt gac ctg 962 Thr Thr Trp Phe Leu Leu Asp Val Ile AlaAla Leu Pro Phe Asp Leu 305 310 315 cta cat gcc ttc aag gtc aac gtg tacttc ggg gcc cat ctg ctg aag 1010 Leu His Ala Phe Lys Val Asn Val Tyr PheGly Ala His Leu Leu Lys 320 325 330 335 acg gtg cgc ctg ctg cgc ctg ctgcgc ctg ctt ccg cgg ctg gac cgg 1058 Thr Val Arg Leu Leu Arg Leu Leu ArgLeu Leu Pro Arg Leu Asp Arg 340 345 350 tac tcg cag tac agc gcc gtg gtgctg aca ctg ctc atg gcc gtg ttc 1106 Tyr Ser Gln Tyr Ser Ala Val Val LeuThr Leu Leu Met Ala Val Phe 355 360 365 gcc ctg ctc gcg cac tgg gtc gcctgc gtc tgg ttt tac att ggc cag 1154 Ala Leu Leu Ala His Trp Val Ala CysVal Trp Phe Tyr Ile Gly Gln 370 375 380 cgg gag atc gag agc agc gaa tccgag ctg cct gag att ggc tgg ctg 1202 Arg Glu Ile Glu Ser Ser Glu Ser GluLeu Pro Glu Ile Gly Trp Leu 385 390 395 cag gag ctg gcc cgc cga ctg gagact ccc tac tac ctg gtg ggc cgg 1250 Gln Glu Leu Ala Arg Arg Leu Glu ThrPro Tyr Tyr Leu Val Gly Arg 400 405 410 415 agg cca gct gga ggg aac agctcc ggc cag agt gac aac tgc agc agc 1298 Arg Pro Ala Gly Gly Asn Ser SerGly Gln Ser Asp Asn Cys Ser Ser 420 425 430 agc agc gag gcc aac ggg acgggg ctg gag ctg ctg ggc ggc ccg tcg 1346 Ser Ser Glu Ala Asn Gly Thr GlyLeu Glu Leu Leu Gly Gly Pro Ser 435 440 445 ctg cgc agc gcc tac atc acctcc ctc tac ttc gca ctc agc agc ctc 1394 Leu Arg Ser Ala Tyr Ile Thr SerLeu Tyr Phe Ala Leu Ser Ser Leu 450 455 460 acc agc gtg ggc ttc ggc aacgtg tcc gcc aac acg gac acc gag aag 1442 Thr Ser Val Gly Phe Gly Asn ValSer Ala Asn Thr Asp Thr Glu Lys 465 470 475 atc ttc tcc atc tgc acc atgctc atc ggc gcc ctg atg cac gcg gtg 1490 Ile Phe Ser Ile Cys Thr Met LeuIle Gly Ala Leu Met His Ala Val 480 485 490 495 gtg ttt ggg aac gtg acggcc atc atc cag cgc atg tac gcc cgc cgc 1538 Val Phe Gly Asn Val Thr AlaIle Ile Gln Arg Met Tyr Ala Arg Arg 500 505 510 ttt ctg tac cac agc cgcacg cgc gac ctg cgc gac tac atc cgc atc 1586 Phe Leu Tyr His Ser Arg ThrArg Asp Leu Arg Asp Tyr Ile Arg Ile 515 520 525 cac cgt atc ccc aag cccctc aag cag cgc atg ctg gag tac ttc cag 1634 His Arg Ile Pro Lys Pro LeuLys Gln Arg Met Leu Glu Tyr Phe Gln 530 535 540 gcc acc tgg gcg gtg aacaat ggc atc gac acc acc gag ctg ctg cag 1682 Ala Thr Trp Ala Val Asn AsnGly Ile Asp Thr Thr Glu Leu Leu Gln 545 550 555 agc ctc cct gac gag ctgcgc gca gac atc gcc atg cac ctg cac aag 1730 Ser Leu Pro Asp Glu Leu ArgAla Asp Ile Ala Met His Leu His Lys 560 565 570 575 gag gtc ctg cag ctgcca ctg ttt gag gcg gcc agc cgc ggc tgc ctg 1778 Glu Val Leu Gln Leu ProLeu Phe Glu Ala Ala Ser Arg Gly Cys Leu 580 585 590 cgg gca ctg tct ctggcc ctg cgg ccc gcc ttc tgc acg ccg ggc gag 1826 Arg Ala Leu Ser Leu AlaLeu Arg Pro Ala Phe Cys Thr Pro Gly Glu 595 600 605 tac ctc atc cac caaggc gat gcc ctg cag gcc ctc tac ttt gtc tgc 1874 Tyr Leu Ile His Gln GlyAsp Ala Leu Gln Ala Leu Tyr Phe Val Cys 610 615 620 tct ggc tcc atg gaggtg ctc aag ggt ggc acc gtg ctc gcc atc cta 1922 Ser Gly Ser Met Glu ValLeu Lys Gly Gly Thr Val Leu Ala Ile Leu 625 630 635 ggg aag ggc gac ctgatc ggc tgt gag ctg ccc cgg cgg gag cag gtg 1970 Gly Lys Gly Asp Leu IleGly Cys Glu Leu Pro Arg Arg Glu Gln Val 640 645 650 655 gta aag gcc aatgcc gac gtg aag ggg ctg acg tac tgc gtc ctg cag 2018 Val Lys Ala Asn AlaAsp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln 660 665 670 tgt ctg cag ctggct ggc ctg cac gac agc ctt gcg ctg tac ccc gag 2066 Cys Leu Gln Leu AlaGly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu 675 680 685 ttt gcc ccg cgcttc agt cgt ggc ctc cga ggg gag ctc agc tac aac 2114 Phe Ala Pro Arg PheSer Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn 690 695 700 ctg ggt gct ggggga ggc tct gca gag gtg gac acc agc tcc ctg agc 2162 Leu Gly Ala Gly GlyGly Ser Ala Glu Val Asp Thr Ser Ser Leu Ser 705 710 715 ggc gac aat accctt atg tcc acg ctg gag gag aag gag aca gat ggg 2210 Gly Asp Asn Thr LeuMet Ser Thr Leu Glu Glu Lys Glu Thr Asp Gly 720 725 730 735 gag cag ggcccc acg gtc tcc cca gcc cca gct gat gag ccc tcc agc 2258 Glu Gln Gly ProThr Val Ser Pro Ala Pro Ala Asp Glu Pro Ser Ser 740 745 750 ccc ctg ctgtcc cct ggc tgc acc tcc tca tcc tca gct gcc aag ctg 2306 Pro Leu Leu SerPro Gly Cys Thr Ser Ser Ser Ser Ala Ala Lys Leu 755 760 765 cta tcc ccacgt cga aca gca ccc cgg cct cgt cta ggt ggc aga ggg 2354 Leu Ser Pro ArgArg Thr Ala Pro Arg Pro Arg Leu Gly Gly Arg Gly 770 775 780 agg cca ggcagg gca ggg gct ttg aag gct gag gct ggc ccc tct gct 2402 Arg Pro Gly ArgAla Gly Ala Leu Lys Ala Glu Ala Gly Pro Ser Ala 785 790 795 ccc cca cgggcc cta gag ggg cta cgg ctg ccc ccc atg cca tgg aat 2450 Pro Pro Arg AlaLeu Glu Gly Leu Arg Leu Pro Pro Met Pro Trp Asn 800 805 810 815 gtg ccccca gat ctg agc ccc agg gta gta gat ggc att gaa gac ggc 2498 Val Pro ProAsp Leu Ser Pro Arg Val Val Asp Gly Ile Glu Asp Gly 820 825 830 tgt ggctcg gac cag ccc aag ttc tct ttc cgc gtg ggc cag tct ggc 2546 Cys Gly SerAsp Gln Pro Lys Phe Ser Phe Arg Val Gly Gln Ser Gly 835 840 845 ccg gaatgt agc agc agc ccc tcc cct gga cca gag agc ggc ctg ctc 2594 Pro Glu CysSer Ser Ser Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu 850 855 860 act gttccc cat ggg ccc agc gag gca agg aac aca gac aca ctg gac 2642 Thr Val ProHis Gly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp 865 870 875 aag cttcgg cag gcg gtg aca gag ctg tca gag cag gtg ctg cag atg 2690 Lys Leu ArgGln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met 880 885 890 895 cgggaa gga ctg cag tca ctt cgc cag gct gtg cag ctt gtc ctg gcg 2738 Arg GluGly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala 900 905 910 ccccac agg gag ggt ccg tgc cct cgg gca tcg gga gag ggg ccg tgc 2786 Pro HisArg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys 915 920 925 ccagcc agc acc tcc ggg ctt ctg cag cct ctg tgt gtg gac act ggg 2834 Pro AlaSer Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly 930 935 940 gcatcc tcc tac tgc ctg cag ccc cca gct ggc tct gtc ttg agt ggg 2882 Ala SerSer Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser Gly 945 950 955 acttgg ccc cac cct cgt ccg ggg cct cct ccc ctc atg gca ccc tgg 2930 Thr TrpPro His Pro Arg Pro Gly Pro Pro Pro Leu Met Ala Pro Trp 960 965 970 975ccc tgg ggt ccc cca gcg tct cag agc tcc ccc tgg cct cga gcc aca 2978 ProTrp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp Pro Arg Ala Thr 980 985 990gct ttc tgg acc tcc acc tca gac tca gag ccc cct gcc tca gga gac 3026 AlaPhe Trp Thr Ser Thr Ser Asp Ser Glu Pro Pro Ala Ser Gly Asp 995 10001005 ctc tgc tct gag ccc agc acc cct gcc tcc cct cct cct tct gag gaa3074 Leu Cys Ser Glu Pro Ser Thr Pro Ala Ser Pro Pro Pro Ser Glu Glu1010 1015 1020 ggg gct agg act ggg ccc gca gag cct gtg agc cag gct gaggct acc 3122 Gly Ala Arg Thr Gly Pro Ala Glu Pro Val Ser Gln Ala Glu AlaThr 1025 1030 1035 agc act gga gag ccc cca cca ggg tca ggg ggc ctg gccttg ccc tgg 3170 Ser Thr Gly Glu Pro Pro Pro Gly Ser Gly Gly Leu Ala LeuPro Trp 1040 1045 1050 1055 gac ccc cac agc ctg gag atg gtg ctt att ggctgc cat ggc tct ggc 3218 Asp Pro His Ser Leu Glu Met Val Leu Ile Gly CysHis Gly Ser Gly 1060 1065 1070 aca gtc cag tgg acc cag gaa gaa ggc acaggg gtc tga gtaccagccc 3267 Thr Val Gln Trp Thr Gln Glu Glu Gly Thr GlyVal 1075 1080 tagaactcag cgttgccagg tgtgctgcca tctgctgttc ggcccaacctcagagt 3323 2 1083 PRT Homo sapiens 2 Met Pro Ala Met Arg Gly Leu LeuAla Pro Gln Asn Thr Phe Leu Asp 1 5 10 15 Thr Ile Ala Thr Arg Phe AspGly Thr His Ser Asn Phe Val Leu Gly 20 25 30 Asn Ala Gln Val Ala Gly LeuPhe Pro Val Val Tyr Cys Ser Asp Gly 35 40 45 Phe Cys Asp Leu Thr Gly PheSer Arg Ala Glu Val Met Gln Arg Gly 50 55 60 Cys Ala Cys Ser Phe Leu TyrGly Pro Asp Thr Ser Glu Leu Val Arg 65 70 75 80 Gln Gln Ile Arg Lys AlaLeu Asp Glu His Lys Glu Phe Lys Ala Glu 85 90 95 Leu Ile Leu Tyr Arg LysSer Gly Leu Pro Phe Trp Cys Leu Leu Asp 100 105 110 Val Ile Pro Ile LysAsn Glu Lys Gly Glu Val Ala Leu Phe Leu Val 115 120 125 Ser His Lys AspIle Ser Glu Thr Lys Asn Arg Gly Gly Pro Asp Arg 130 135 140 Trp Lys GluThr Gly Gly Gly Arg Arg Arg Tyr Gly Arg Ala Arg Ser 145 150 155 160 LysGly Phe Asn Ala Asn Arg Arg Arg Ser Arg Ala Val Leu Tyr His 165 170 175Leu Ser Gly His Leu Gln Lys Gln Pro Lys Gly Lys His Lys Leu Asn 180 185190 Lys Gly Val Phe Gly Glu Lys Pro Asn Leu Pro Glu Tyr Lys Val Ala 195200 205 Ala Ile Arg Lys Ser Pro Phe Ile Leu Leu His Cys Gly Ala Leu Arg210 215 220 Ala Thr Trp Asp Gly Phe Ile Leu Leu Ala Thr Leu Tyr Val AlaVal 225 230 235 240 Thr Val Pro Tyr Ser Val Cys Val Ser Thr Ala Arg GluPro Ser Ala 245 250 255 Ala Arg Gly Pro Pro Ser Val Cys Asp Leu Ala ValGlu Val Leu Phe 260 265 270 Ile Leu Asp Ile Val Leu Asn Phe Arg Thr ThrPhe Val Ser Lys Ser 275 280 285 Gly Gln Val Val Phe Ala Pro Lys Ser IleCys Leu His Tyr Val Thr 290 295 300 Thr Trp Phe Leu Leu Asp Val Ile AlaAla Leu Pro Phe Asp Leu Leu 305 310 315 320 His Ala Phe Lys Val Asn ValTyr Phe Gly Ala His Leu Leu Lys Thr 325 330 335 Val Arg Leu Leu Arg LeuLeu Arg Leu Leu Pro Arg Leu Asp Arg Tyr 340 345 350 Ser Gln Tyr Ser AlaVal Val Leu Thr Leu Leu Met Ala Val Phe Ala 355 360 365 Leu Leu Ala HisTrp Val Ala Cys Val Trp Phe Tyr Ile Gly Gln Arg 370 375 380 Glu Ile GluSer Ser Glu Ser Glu Leu Pro Glu Ile Gly Trp Leu Gln 385 390 395 400 GluLeu Ala Arg Arg Leu Glu Thr Pro Tyr Tyr Leu Val Gly Arg Arg 405 410 415Pro Ala Gly Gly Asn Ser Ser Gly Gln Ser Asp Asn Cys Ser Ser Ser 420 425430 Ser Glu Ala Asn Gly Thr Gly Leu Glu Leu Leu Gly Gly Pro Ser Leu 435440 445 Arg Ser Ala Tyr Ile Thr Ser Leu Tyr Phe Ala Leu Ser Ser Leu Thr450 455 460 Ser Val Gly Phe Gly Asn Val Ser Ala Asn Thr Asp Thr Glu LysIle 465 470 475 480 Phe Ser Ile Cys Thr Met Leu Ile Gly Ala Leu Met HisAla Val Val 485 490 495 Phe Gly Asn Val Thr Ala Ile Ile Gln Arg Met TyrAla Arg Arg Phe 500 505 510 Leu Tyr His Ser Arg Thr Arg Asp Leu Arg AspTyr Ile Arg Ile His 515 520 525 Arg Ile Pro Lys Pro Leu Lys Gln Arg MetLeu Glu Tyr Phe Gln Ala 530 535 540 Thr Trp Ala Val Asn Asn Gly Ile AspThr Thr Glu Leu Leu Gln Ser 545 550 555 560 Leu Pro Asp Glu Leu Arg AlaAsp Ile Ala Met His Leu His Lys Glu 565 570 575 Val Leu Gln Leu Pro LeuPhe Glu Ala Ala Ser Arg Gly Cys Leu Arg 580 585 590 Ala Leu Ser Leu AlaLeu Arg Pro Ala Phe Cys Thr Pro Gly Glu Tyr 595 600 605 Leu Ile His GlnGly Asp Ala Leu Gln Ala Leu Tyr Phe Val Cys Ser 610 615 620 Gly Ser MetGlu Val Leu Lys Gly Gly Thr Val Leu Ala Ile Leu Gly 625 630 635 640 LysGly Asp Leu Ile Gly Cys Glu Leu Pro Arg Arg Glu Gln Val Val 645 650 655Lys Ala Asn Ala Asp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln Cys 660 665670 Leu Gln Leu Ala Gly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu Phe 675680 685 Ala Pro Arg Phe Ser Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn Leu690 695 700 Gly Ala Gly Gly Gly Ser Ala Glu Val Asp Thr Ser Ser Leu SerGly 705 710 715 720 Asp Asn Thr Leu Met Ser Thr Leu Glu Glu Lys Glu ThrAsp Gly Glu 725 730 735 Gln Gly Pro Thr Val Ser Pro Ala Pro Ala Asp GluPro Ser Ser Pro 740 745 750 Leu Leu Ser Pro Gly Cys Thr Ser Ser Ser SerAla Ala Lys Leu Leu 755 760 765 Ser Pro Arg Arg Thr Ala Pro Arg Pro ArgLeu Gly Gly Arg Gly Arg 770 775 780 Pro Gly Arg Ala Gly Ala Leu Lys AlaGlu Ala Gly Pro Ser Ala Pro 785 790 795 800 Pro Arg Ala Leu Glu Gly LeuArg Leu Pro Pro Met Pro Trp Asn Val 805 810 815 Pro Pro Asp Leu Ser ProArg Val Val Asp Gly Ile Glu Asp Gly Cys 820 825 830 Gly Ser Asp Gln ProLys Phe Ser Phe Arg Val Gly Gln Ser Gly Pro 835 840 845 Glu Cys Ser SerSer Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu Thr 850 855 860 Val Pro HisGly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp Lys 865 870 875 880 LeuArg Gln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met Arg 885 890 895Glu Gly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala Pro 900 905910 His Arg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys Pro 915920 925 Ala Ser Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly Ala930 935 940 Ser Ser Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser GlyThr 945 950 955 960 Trp Pro His Pro Arg Pro Gly Pro Pro Pro Leu Met AlaPro Trp Pro 965 970 975 Trp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp ProArg Ala Thr Ala 980 985 990 Phe Trp Thr Ser Thr Ser Asp Ser Glu Pro ProAla Ser Gly Asp Leu 995 1000 1005 Cys Ser Glu Pro Ser Thr Pro Ala SerPro Pro Pro Ser Glu Glu Gly 1010 1015 1020 Ala Arg Thr Gly Pro Ala GluPro Val Ser Gln Ala Glu Ala Thr Ser 1025 1030 1035 1040 Thr Gly Glu ProPro Pro Gly Ser Gly Gly Leu Ala Leu Pro Trp Asp 1045 1050 1055 Pro HisSer Leu Glu Met Val Leu Ile Gly Cys His Gly Ser Gly Thr 1060 1065 1070Val Gln Trp Thr Gln Glu Glu Gly Thr Gly Val 1075 1080 3 26 DNAArtificial Sequence Description of Artificial SequencePrimer 3ggaattccct aagatgccgg ccatgc 26 4 30 DNA Artificial Sequence Descriptionof Artificial SequencePrimer 4 gctctagagc actctgaggt tgggccgaac 30 53064 DNA Homo sapiens CDS (4)..(3057) 5 gcc atg ccg gtc atg aag ggg ttgctg gcc ccg caa aac acc ttc ctg 48 Met Pro Val Met Lys Gly Leu Leu AlaPro Gln Asn Thr Phe Leu 1 5 10 15 gac acc atc gcc acc cgt ttt gac ggaacg cac agc aac ttc ctg ctg 96 Asp Thr Ile Ala Thr Arg Phe Asp Gly ThrHis Ser Asn Phe Leu Leu 20 25 30 gcc aac gca cag ggc aca cgg ggc ttt cccatc gtc tac tgc tcc gac 144 Ala Asn Ala Gln Gly Thr Arg Gly Phe Pro IleVal Tyr Cys Ser Asp 35 40 45 ggc ttc tgc gag ctc aca ggc tac ggt cgc accgag gtc atg cag aag 192 Gly Phe Cys Glu Leu Thr Gly Tyr Gly Arg Thr GluVal Met Gln Lys 50 55 60 acc tgc agc tgc cgt ttc ctc tac ggc cca gag accagt gag cca gcc 240 Thr Cys Ser Cys Arg Phe Leu Tyr Gly Pro Glu Thr SerGlu Pro Ala 65 70 75 ctg cag cgt ctg cac aaa gcc ctg gag ggc cac cag gagcac cgg gct 288 Leu Gln Arg Leu His Lys Ala Leu Glu Gly His Gln Glu HisArg Ala 80 85 90 95 gaa atc tgc ttc tac cgc aag gat ggc tca gcc ttt tggtgc ctc ctg 336 Glu Ile Cys Phe Tyr Arg Lys Asp Gly Ser Ala Phe Trp CysLeu Leu 100 105 110 gac atg atg ccc atc aag aat gag atg ggg gag gtc gtgctg ttc ctc 384 Asp Met Met Pro Ile Lys Asn Glu Met Gly Glu Val Val LeuPhe Leu 115 120 125 ttt tcc ttc aag gat atc act cag agt gga agc cca ggactt ggc ccc 432 Phe Ser Phe Lys Asp Ile Thr Gln Ser Gly Ser Pro Gly LeuGly Pro 130 135 140 caa gga ggc cgc ggg gac agt aat cac gaa aac tcc cttggt aga agg 480 Gln Gly Gly Arg Gly Asp Ser Asn His Glu Asn Ser Leu GlyArg Arg 145 150 155 gga gcc acc tgg aaa ttt cgg tct gcc aga aga cgg agccgt act gtc 528 Gly Ala Thr Trp Lys Phe Arg Ser Ala Arg Arg Arg Ser ArgThr Val 160 165 170 175 cta cac cga ctg acc ggc cac ttt ggc cgc cgg ggccag gga ggc atg 576 Leu His Arg Leu Thr Gly His Phe Gly Arg Arg Gly GlnGly Gly Met 180 185 190 aag gcc aat aat aac gtg ttt gag cca aag cca tcagtg ccc gag tac 624 Lys Ala Asn Asn Asn Val Phe Glu Pro Lys Pro Ser ValPro Glu Tyr 195 200 205 aag gtg gcc tcc gtg ggg ggg tct cgc tgc ctc ctcctc cac tac agc 672 Lys Val Ala Ser Val Gly Gly Ser Arg Cys Leu Leu LeuHis Tyr Ser 210 215 220 gtc tcc aag gcc atc tgg gac ggc ctt atc ctc cttgcc acc ttc tac 720 Val Ser Lys Ala Ile Trp Asp Gly Leu Ile Leu Leu AlaThr Phe Tyr 225 230 235 gtt gcg gtc acc gtc ccc tac aat gtc tgt ttc tcgggt gac gat gac 768 Val Ala Val Thr Val Pro Tyr Asn Val Cys Phe Ser GlyAsp Asp Asp 240 245 250 255 acc ccc atc act tcg cga cac acc ctt gtc agcgac atc gcc gtg gaa 816 Thr Pro Ile Thr Ser Arg His Thr Leu Val Ser AspIle Ala Val Glu 260 265 270 atg ctc ttc atc cta gat atc atc ctg aac ttccgc acc acc tat gtg 864 Met Leu Phe Ile Leu Asp Ile Ile Leu Asn Phe ArgThr Thr Tyr Val 275 280 285 tcc cag tcc ggc cag gta atc tct gct cct cgttcc att ggc ctc cac 912 Ser Gln Ser Gly Gln Val Ile Ser Ala Pro Arg SerIle Gly Leu His 290 295 300 tac ctg gcc acc tgg ttc ttc atc gac ctt attgct gct ctg ccc ttt 960 Tyr Leu Ala Thr Trp Phe Phe Ile Asp Leu Ile AlaAla Leu Pro Phe 305 310 315 gac ctg ctt tac atc ttc aac atc acc gtg acctcg ctg gtg cac cta 1008 Asp Leu Leu Tyr Ile Phe Asn Ile Thr Val Thr SerLeu Val His Leu 320 325 330 335 ctg aag aca gtg cgg ctg ttg cgg ctg ctgcgg ctg ctg cag aag ctg 1056 Leu Lys Thr Val Arg Leu Leu Arg Leu Leu ArgLeu Leu Gln Lys Leu 340 345 350 gag cgg tac tct cag tgc agt gct gtg gtgctc acg ctg ctc atg tcg 1104 Glu Arg Tyr Ser Gln Cys Ser Ala Val Val LeuThr Leu Leu Met Ser 355 360 365 gtc ttt gcg ctc ctt gcc cac tgg atg gcctgc atc tgg tat gtc atc 1152 Val Phe Ala Leu Leu Ala His Trp Met Ala CysIle Trp Tyr Val Ile 370 375 380 ggg cgc cgg gag atg gag gcc aat gac ccgctg ctc tgg gac att ggc 1200 Gly Arg Arg Glu Met Glu Ala Asn Asp Pro LeuLeu Trp Asp Ile Gly 385 390 395 tgg ttg cat gag ttg ggc aag cgt ctg gaggtg ccc tat gtc aat ggc 1248 Trp Leu His Glu Leu Gly Lys Arg Leu Glu ValPro Tyr Val Asn Gly 400 405 410 415 tcg gtg ggc ggc cca tca cgg cgc agcgcc tac atc gcg gca ctg tac 1296 Ser Val Gly Gly Pro Ser Arg Arg Ser AlaTyr Ile Ala Ala Leu Tyr 420 425 430 ttc act cta agc agc ctc acc agt gtgggc ttt ggc aac gtg tgt gcc 1344 Phe Thr Leu Ser Ser Leu Thr Ser Val GlyPhe Gly Asn Val Cys Ala 435 440 445 aac acc gac gcg gag aag atc ttc tccatc tgc acg atg ctc ata ggc 1392 Asn Thr Asp Ala Glu Lys Ile Phe Ser IleCys Thr Met Leu Ile Gly 450 455 460 gcc ctg atg cac gct gtg gtg ttc gggaac gtg aca gcc atc atc cag 1440 Ala Leu Met His Ala Val Val Phe Gly AsnVal Thr Ala Ile Ile Gln 465 470 475 cgc atg tac tcg cgc cgc tcg ctc taccac agc cgc atg aag gac ctc 1488 Arg Met Tyr Ser Arg Arg Ser Leu Tyr HisSer Arg Met Lys Asp Leu 480 485 490 495 aag gac ttc atc cgt gtg cac cgcctg ccg cgg ccg ctc aag cag cgc 1536 Lys Asp Phe Ile Arg Val His Arg LeuPro Arg Pro Leu Lys Gln Arg 500 505 510 atg ctc gaa tac ttc cag acc acgtgg gcc gtc aac agc ggc atc gac 1584 Met Leu Glu Tyr Phe Gln Thr Thr TrpAla Val Asn Ser Gly Ile Asp 515 520 525 gcc aac gag tta ctg cgt gac ttccca gac gag ctg aga gct gac att 1632 Ala Asn Glu Leu Leu Arg Asp Phe ProAsp Glu Leu Arg Ala Asp Ile 530 535 540 gct atg cac ctg aat cgg gag atcctg cag ctg ccg ttg ttc ggg gca 1680 Ala Met His Leu Asn Arg Glu Ile LeuGln Leu Pro Leu Phe Gly Ala 545 550 555 gcg agc agg ggc tgc ctg cgg gcccta tcg ctg cac atc aag acc tcg 1728 Ala Ser Arg Gly Cys Leu Arg Ala LeuSer Leu His Ile Lys Thr Ser 560 565 570 575 ttc tgc gct ccg ggc gag tacctg ttg cgc cgt ggg gat gcc ctg cag 1776 Phe Cys Ala Pro Gly Glu Tyr LeuLeu Arg Arg Gly Asp Ala Leu Gln 580 585 590 gca cat tac tat gtc tgc tccggc tcg ctt gag gtg ctc cga gac aac 1824 Ala His Tyr Tyr Val Cys Ser GlySer Leu Glu Val Leu Arg Asp Asn 595 600 605 atg gtg ctg gcc atc ctg gggaag ggg gac ctg att gga gca gat atc 1872 Met Val Leu Ala Ile Leu Gly LysGly Asp Leu Ile Gly Ala Asp Ile 610 615 620 cct gag ccg ggg cag gag cctggg ttg gga gca gac cca aac ttc gtg 1920 Pro Glu Pro Gly Gln Glu Pro GlyLeu Gly Ala Asp Pro Asn Phe Val 625 630 635 cta aag acc agt gct gat gtgaaa gct ctg acc tac tgt ggc ctg cag 1968 Leu Lys Thr Ser Ala Asp Val LysAla Leu Thr Tyr Cys Gly Leu Gln 640 645 650 655 cag ctg agc agc cga gggctg gct gag gtc ctg agg ctc tat cct gag 2016 Gln Leu Ser Ser Arg Gly LeuAla Glu Val Leu Arg Leu Tyr Pro Glu 660 665 670 tat ggg gct gcc ttc cgggct ggc ctg ccc cgg gac ctc acc ttc aac 2064 Tyr Gly Ala Ala Phe Arg AlaGly Leu Pro Arg Asp Leu Thr Phe Asn 675 680 685 ctg cgc cag ggc tct gacacc agt ggc ctc agc cgc ttt tcc cga tcc 2112 Leu Arg Gln Gly Ser Asp ThrSer Gly Leu Ser Arg Phe Ser Arg Ser 690 695 700 cct cgc ctc tcc cag ccccgc tca gaa agc ctc ggc tcc tcc tca gac 2160 Pro Arg Leu Ser Gln Pro ArgSer Glu Ser Leu Gly Ser Ser Ser Asp 705 710 715 aag acg ctg cca tcc atcaca gag gcc gag agt ggc gcg gag cct ggg 2208 Lys Thr Leu Pro Ser Ile ThrGlu Ala Glu Ser Gly Ala Glu Pro Gly 720 725 730 735 ggt ggt ccc agg ccccga cgg ccc ctc ctg ctg ccc aac ctc agc cca 2256 Gly Gly Pro Arg Pro ArgArg Pro Leu Leu Leu Pro Asn Leu Ser Pro 740 745 750 gca cgg cct cgg ggctcc ctg gtc agc ctt ttg ggc gag gag ctg ccc 2304 Ala Arg Pro Arg Gly SerLeu Val Ser Leu Leu Gly Glu Glu Leu Pro 755 760 765 cca ttc tca gcc cttgtc tcc tct cct tcc tta tcc cca tcc ctg tcc 2352 Pro Phe Ser Ala Leu ValSer Ser Pro Ser Leu Ser Pro Ser Leu Ser 770 775 780 cct gcc ctg gct ggccag ggc cac agt gcc tcc cct cac ggc ccc ccc 2400 Pro Ala Leu Ala Gly GlnGly His Ser Ala Ser Pro His Gly Pro Pro 785 790 795 agg tgc tct gct gcctgg aag ccc cct cag ctt ctc att ccc cca ctg 2448 Arg Cys Ser Ala Ala TrpLys Pro Pro Gln Leu Leu Ile Pro Pro Leu 800 805 810 815 gga acc ttt ggacct ccg gac ctc agt ccc cgg ata gtg gat ggc att 2496 Gly Thr Phe Gly ProPro Asp Leu Ser Pro Arg Ile Val Asp Gly Ile 820 825 830 gag gac tct ggcagc aca gct gag gcc cct tca ttc cga ttc agc agg 2544 Glu Asp Ser Gly SerThr Ala Glu Ala Pro Ser Phe Arg Phe Ser Arg 835 840 845 agg cct gaa ctgcca agg ccc cgc tcc cag gcg ccc cct aca ggg acc 2592 Arg Pro Glu Leu ProArg Pro Arg Ser Gln Ala Pro Pro Thr Gly Thr 850 855 860 agg ccc agc ccagaa ttg gcc agt gag gct gag gag gtg aag gaa aag 2640 Arg Pro Ser Pro GluLeu Ala Ser Glu Ala Glu Glu Val Lys Glu Lys 865 870 875 gtt tgc cgg ctgaac cag gag atc tct cgt ctc aat cag gag gtg tct 2688 Val Cys Arg Leu AsnGln Glu Ile Ser Arg Leu Asn Gln Glu Val Ser 880 885 890 895 cag ctt agccgg gag ctg cgg cac atc atg ggc ctg ctg cag gcc agg 2736 Gln Leu Ser ArgGlu Leu Arg His Ile Met Gly Leu Leu Gln Ala Arg 900 905 910 ctg ggt ccccca ggc cac cca gca ggc tcc gct tgg acc cca gac cct 2784 Leu Gly Pro ProGly His Pro Ala Gly Ser Ala Trp Thr Pro Asp Pro 915 920 925 cct tgt ccacag ctg agg cca cca tgc ctc tct cct tgt gcg tcc aga 2832 Pro Cys Pro GlnLeu Arg Pro Pro Cys Leu Ser Pro Cys Ala Ser Arg 930 935 940 cca cca cccagc ctc cag gat act acg ctt gct gaa gtt cac tgc cca 2880 Pro Pro Pro SerLeu Gln Asp Thr Thr Leu Ala Glu Val His Cys Pro 945 950 955 gcc agt gtgggg acc atg gag aca ggg act gcg ctc ctg gac ttg aga 2928 Ala Ser Val GlyThr Met Glu Thr Gly Thr Ala Leu Leu Asp Leu Arg 960 965 970 975 cct tccata ttg ccc ccc tac ccc tca gag cct gac cct ctg gga ccc 2976 Pro Ser IleLeu Pro Pro Tyr Pro Ser Glu Pro Asp Pro Leu Gly Pro 980 985 990 tct ccagtg cca gag gcc tca ccc cca acc cca agc ctc ttg agg cac 3024 Ser Pro ValPro Glu Ala Ser Pro Pro Thr Pro Ser Leu Leu Arg His 995 1000 1005 agtttc cag tcc agg tca gac aca ttc cac tga ccctggc 3064 Ser Phe Gln Ser ArgSer Asp Thr Phe His 1010 1015 6 1017 PRT Homo sapiens 6 Met Pro Val MetLys Gly Leu Leu Ala Pro Gln Asn Thr Phe Leu Asp 1 5 10 15 Thr Ile AlaThr Arg Phe Asp Gly Thr His Ser Asn Phe Leu Leu Ala 20 25 30 Asn Ala GlnGly Thr Arg Gly Phe Pro Ile Val Tyr Cys Ser Asp Gly 35 40 45 Phe Cys GluLeu Thr Gly Tyr Gly Arg Thr Glu Val Met Gln Lys Thr 50 55 60 Cys Ser CysArg Phe Leu Tyr Gly Pro Glu Thr Ser Glu Pro Ala Leu 65 70 75 80 Gln ArgLeu His Lys Ala Leu Glu Gly His Gln Glu His Arg Ala Glu 85 90 95 Ile CysPhe Tyr Arg Lys Asp Gly Ser Ala Phe Trp Cys Leu Leu Asp 100 105 110 MetMet Pro Ile Lys Asn Glu Met Gly Glu Val Val Leu Phe Leu Phe 115 120 125Ser Phe Lys Asp Ile Thr Gln Ser Gly Ser Pro Gly Leu Gly Pro Gln 130 135140 Gly Gly Arg Gly Asp Ser Asn His Glu Asn Ser Leu Gly Arg Arg Gly 145150 155 160 Ala Thr Trp Lys Phe Arg Ser Ala Arg Arg Arg Ser Arg Thr ValLeu 165 170 175 His Arg Leu Thr Gly His Phe Gly Arg Arg Gly Gln Gly GlyMet Lys 180 185 190 Ala Asn Asn Asn Val Phe Glu Pro Lys Pro Ser Val ProGlu Tyr Lys 195 200 205 Val Ala Ser Val Gly Gly Ser Arg Cys Leu Leu LeuHis Tyr Ser Val 210 215 220 Ser Lys Ala Ile Trp Asp Gly Leu Ile Leu LeuAla Thr Phe Tyr Val 225 230 235 240 Ala Val Thr Val Pro Tyr Asn Val CysPhe Ser Gly Asp Asp Asp Thr 245 250 255 Pro Ile Thr Ser Arg His Thr LeuVal Ser Asp Ile Ala Val Glu Met 260 265 270 Leu Phe Ile Leu Asp Ile IleLeu Asn Phe Arg Thr Thr Tyr Val Ser 275 280 285 Gln Ser Gly Gln Val IleSer Ala Pro Arg Ser Ile Gly Leu His Tyr 290 295 300 Leu Ala Thr Trp PhePhe Ile Asp Leu Ile Ala Ala Leu Pro Phe Asp 305 310 315 320 Leu Leu TyrIle Phe Asn Ile Thr Val Thr Ser Leu Val His Leu Leu 325 330 335 Lys ThrVal Arg Leu Leu Arg Leu Leu Arg Leu Leu Gln Lys Leu Glu 340 345 350 ArgTyr Ser Gln Cys Ser Ala Val Val Leu Thr Leu Leu Met Ser Val 355 360 365Phe Ala Leu Leu Ala His Trp Met Ala Cys Ile Trp Tyr Val Ile Gly 370 375380 Arg Arg Glu Met Glu Ala Asn Asp Pro Leu Leu Trp Asp Ile Gly Trp 385390 395 400 Leu His Glu Leu Gly Lys Arg Leu Glu Val Pro Tyr Val Asn GlySer 405 410 415 Val Gly Gly Pro Ser Arg Arg Ser Ala Tyr Ile Ala Ala LeuTyr Phe 420 425 430 Thr Leu Ser Ser Leu Thr Ser Val Gly Phe Gly Asn ValCys Ala Asn 435 440 445 Thr Asp Ala Glu Lys Ile Phe Ser Ile Cys Thr MetLeu Ile Gly Ala 450 455 460 Leu Met His Ala Val Val Phe Gly Asn Val ThrAla Ile Ile Gln Arg 465 470 475 480 Met Tyr Ser Arg Arg Ser Leu Tyr HisSer Arg Met Lys Asp Leu Lys 485 490 495 Asp Phe Ile Arg Val His Arg LeuPro Arg Pro Leu Lys Gln Arg Met 500 505 510 Leu Glu Tyr Phe Gln Thr ThrTrp Ala Val Asn Ser Gly Ile Asp Ala 515 520 525 Asn Glu Leu Leu Arg AspPhe Pro Asp Glu Leu Arg Ala Asp Ile Ala 530 535 540 Met His Leu Asn ArgGlu Ile Leu Gln Leu Pro Leu Phe Gly Ala Ala 545 550 555 560 Ser Arg GlyCys Leu Arg Ala Leu Ser Leu His Ile Lys Thr Ser Phe 565 570 575 Cys AlaPro Gly Glu Tyr Leu Leu Arg Arg Gly Asp Ala Leu Gln Ala 580 585 590 HisTyr Tyr Val Cys Ser Gly Ser Leu Glu Val Leu Arg Asp Asn Met 595 600 605Val Leu Ala Ile Leu Gly Lys Gly Asp Leu Ile Gly Ala Asp Ile Pro 610 615620 Glu Pro Gly Gln Glu Pro Gly Leu Gly Ala Asp Pro Asn Phe Val Leu 625630 635 640 Lys Thr Ser Ala Asp Val Lys Ala Leu Thr Tyr Cys Gly Leu GlnGln 645 650 655 Leu Ser Ser Arg Gly Leu Ala Glu Val Leu Arg Leu Tyr ProGlu Tyr 660 665 670 Gly Ala Ala Phe Arg Ala Gly Leu Pro Arg Asp Leu ThrPhe Asn Leu 675 680 685 Arg Gln Gly Ser Asp Thr Ser Gly Leu Ser Arg PheSer Arg Ser Pro 690 695 700 Arg Leu Ser Gln Pro Arg Ser Glu Ser Leu GlySer Ser Ser Asp Lys 705 710 715 720 Thr Leu Pro Ser Ile Thr Glu Ala GluSer Gly Ala Glu Pro Gly Gly 725 730 735 Gly Pro Arg Pro Arg Arg Pro LeuLeu Leu Pro Asn Leu Ser Pro Ala 740 745 750 Arg Pro Arg Gly Ser Leu ValSer Leu Leu Gly Glu Glu Leu Pro Pro 755 760 765 Phe Ser Ala Leu Val SerSer Pro Ser Leu Ser Pro Ser Leu Ser Pro 770 775 780 Ala Leu Ala Gly GlnGly His Ser Ala Ser Pro His Gly Pro Pro Arg 785 790 795 800 Cys Ser AlaAla Trp Lys Pro Pro Gln Leu Leu Ile Pro Pro Leu Gly 805 810 815 Thr PheGly Pro Pro Asp Leu Ser Pro Arg Ile Val Asp Gly Ile Glu 820 825 830 AspSer Gly Ser Thr Ala Glu Ala Pro Ser Phe Arg Phe Ser Arg Arg 835 840 845Pro Glu Leu Pro Arg Pro Arg Ser Gln Ala Pro Pro Thr Gly Thr Arg 850 855860 Pro Ser Pro Glu Leu Ala Ser Glu Ala Glu Glu Val Lys Glu Lys Val 865870 875 880 Cys Arg Leu Asn Gln Glu Ile Ser Arg Leu Asn Gln Glu Val SerGln 885 890 895 Leu Ser Arg Glu Leu Arg His Ile Met Gly Leu Leu Gln AlaArg Leu 900 905 910 Gly Pro Pro Gly His Pro Ala Gly Ser Ala Trp Thr ProAsp Pro Pro 915 920 925 Cys Pro Gln Leu Arg Pro Pro Cys Leu Ser Pro CysAla Ser Arg Pro 930 935 940 Pro Pro Ser Leu Gln Asp Thr Thr Leu Ala GluVal His Cys Pro Ala 945 950 955 960 Ser Val Gly Thr Met Glu Thr Gly ThrAla Leu Leu Asp Leu Arg Pro 965 970 975 Ser Ile Leu Pro Pro Tyr Pro SerGlu Pro Asp Pro Leu Gly Pro Ser 980 985 990 Pro Val Pro Glu Ala Ser ProPro Thr Pro Ser Leu Leu Arg His Ser 995 1000 1005 Phe Gln Ser Arg SerAsp Thr Phe His 1010 1015 7 19 DNA Artificial Sequence Description ofArtificial SequencePrimer 7 gccatgccgg tcatgaagg 19 8 20 DNA ArtificialSequence Description of Artificial SequencePrimer 8 gccagggtcagtggaatgtg 20 9 3715 DNA Rattus sp. 9 ctgctggggc ctacgaacct gggccgggcatagccccccg acggctactc tagggggcgc 60 ggggcccggc ggggggcggc cgagccaggcgccctccccc ggcgctgagt ccccgcgccc 120 cggagggatg gggcgggcgg tccccgccgcctaagatgcc ggccatgcgg gggctccttg 180 cgccgcagaa caccttcctg gacaccatcgccacccgctt cgacgggacg cacagtaact 240 tcgtcctggg caacgcccag gtggcagggctcttccctgt ggtctactgc tccgatggct 300 tctgtgacct cacgggtttc tccagagctgaggtcatgca gcgaggctgt gcctgctcct 360 tcctctatgg gccagacacc agtgagttggtccgccaaca gatccgaaaa gccctggatg 420 agcacaaaga attcaaggct gaactgatcctgtaccggaa gagcgggctt ccattctggt 480 gtctcctgga tgtgatacct ataaaaaacgagaaggggga ggtggccctc ttcctggtct 540 ctcacaagga catcagtgag accaagaaccgaggaggccc tgacaactgg aaggagagag 600 gtggtggccg acgcagatat ggtcgggcaggatccaaagg ctttaatgcc aatcggaggc 660 gcagccgggc ggttctctac cacctctctggtcacctgca gaaacaaccc aagggcaagc 720 acaaactcaa taagggtgtg tttggagagaagccaaattt gcccgaatat aaagtcgctg 780 ctatccggaa gtcacccttt atcctgctgcactgtggggc tctgagagcc acctgggatg 840 gcttcatcct gctcgccacg ctctacgtggctgtcactgt gccatacagc gtgtgtgtga 900 gcacagcacg ggagcccagt gctgcccgtggcccacctag tgtctgtgac ctggccgtgg 960 aagtcctctt catcttagat attgtgctgaattttcgtac tacatttgtg tccaagtcag 1020 gccaggtggt attcgcccca aagtccatttgcctccacta cgtcaccacc tggttcctgc 1080 tggatgtcat agcagcactg ccctttgacctactacatgc cttcaaggtc aatgtgtacg 1140 ttggggctca cctactgaag accgtgcggctgcttcggct gctgcgccta ctaccaagac 1200 tggaccggta ctctcagtat agcgctgttgtgctcacctt gctcatggct gtgtttgccc 1260 tgctcgccca ctgggtggcc tgcgtttggttctacatcgg ccagcaagag attgagaaca 1320 gcgagtcaga gctgcctgag atcggctggctgcaggagct ggcacgcagg ctggagacgc 1380 cctattacct ggtgagccgg agtccagatggagggaacag ctctggccag agtgaaaact 1440 gcagtagcag tggcggcggc agcgaagccaacgggactgg gctggagctg ctgggtggcc 1500 catccctacg cagcgcctac atcacctccttgtacttcgc gctcagcagt ctcaccagtg 1560 tgggcttcgg caatgtgtcc gctaacacagacactgagaa gattttctcc atctgcacca 1620 tgcttattgg agctctgatg catgcagtggtgtttgggaa tgtgacagcc atcatccagc 1680 gcatgtacgc tcgccgcttt ctgtaccacagccgcacccg tgacctgcga gactacattc 1740 gcatccaccg catccccaag cccctcaagcagcgcatgct cgagtacttc caagccacct 1800 gggccgtgaa caacggcatc gataccactgagctgctgca gagccttccg gatgagcttc 1860 gagcagacat cgccatgcac ctgcacaaggaggtcctgca gctgccattg ttcgaggcag 1920 cgagccgtgg ctgcctgcgg gcactgtctctggccctgag gcccgccttc tgcacgccgg 1980 gcgagtacct cattcaccaa ggcgatgctctccaggctct ctactttgtg tgctcaggtt 2040 ccatggaggt cctcaaaggt ggcaccgtcctcgccattct agggaagggt gacctgatcg 2100 gctgtgagct gccccagcga gagcaagtagtgaaggccaa tgccgacgta aaggggctga 2160 catactgcgt cctacagtgc ctgcagctggctgggctgca cgagagcctc gcactgtacc 2220 ctgagtttgc cccacgcttt agccgtggcctccgagggga gctcagctac aacctgggag 2280 ctggaggagt gtctgcagag gtggataccagctcactgag tggtgacaac accctcatgt 2340 ccacactgga ggagaaggag acagatggggagcaaggaca cacgatctca ccagccccag 2400 cagatgagcc ctccagcccc ctgctgtcacctggctgtac ctcctcctcc tcagcggcca 2460 aactactctc cccacgtcga actgcaccccggccgaggct gggtggcaga gggcggccaa 2520 gtagggcagg ggttttgaag cctgaggctggtccttctgc tcatccacgg acacttgatg 2580 ggttgcagct gccccccatg ccatggaatgtacctccaga cctgagcccc agggtcgtag 2640 atggcattga ggatggctgc ggctctgaccagcacaagtt ctctttccgg gtgggtcagt 2700 ctggcccaga atgtagcagc agcccctccccaggaacaga gagtggcctg ctcactgtcc 2760 ccttggtgcc cagtgaggca agaaacacagacacactgga caagctacgg caggcggtga 2820 cggagctgtc tgaacaggtg ctgcagatgcgagagggact gcagtcactt cgccaggctg 2880 tgcagctcat cctggtgccc caaggggaaggccagtgtcc ccgggtatca ggagaggggc 2940 catgcccagc cactgcctct gggctcctacaacccctgcg tgtggacact ggggcatcat 3000 cctactgcct gcagccccca gcaggttcagtcttgagtgg gacctggcct cacccccgtc 3060 cagggcatcc ccctcccctc atggcaccctggccctgggg ccccccagca tctcagagct 3120 ccccctggcc tcgagccaca gctttatggacctccacctc agactcagag ccccctggct 3180 ctggagacct ctgctctgag cccagcaccccagcctcacc ccctcctcct gaggaaggag 3240 ctaggactgg gactcctgca cctgtgagccaggctgaggc taccagtact ggagagcccc 3300 ctccggggtc agggggccga gccttgccctgggatcccca cagcctagag atggtgctca 3360 tcggctgcca tggccctggc tcggtccagtggacccagga ggagggcaca ggagtctgac 3420 cacaggcagg gagaggggtt ctgcaaacacccgccacctg ctgaccgccc agcctcacag 3480 ggctgccctc tggctcaggg cagggaacctaaggaaggag gagggtgagc tggagcctca 3540 ggccccaggc cagggatcca cgggttctgctccactgacc tgacccaatg ggggcagagg 3600 cctgaggacg aggaggggtt ctgccattccttgcatgtgc ccatctccac tgtcctctgt 3660 cctcatgttt tttatattaa aaaacataaaaaaaaaccaa taaagaaact acttt 3715 10 3736 DNA Rattus sp. 10 caggcagcggcggcgagagg aggggaggag gcaggccggc gcatggggcg ccccggcccc 60 gccggtagcgcgccccctcc ggccaggccg cgctgaacgc agcccgcgca acgcctcgaa 120 ttcgtacccggggccatgcc ggtcatgaag gggttgctgg ccccgcaaaa cacctttctg 180 gacaccatcgccactcgctt tgacggcacg cacagcaact ttcttctggc caatgcccag 240 ggcccacggggttttcccat cgtctactgc tctgacggct tctgtgagct cacaggctac 300 ggccgcaccgaggtcatgca gaaaacctgt agctgccggt tcctctatgg cccagagacc 360 agtgagccggccttgcaacg gttacaaaaa gccctggagg gccaccaaga acacagagct 420 gaaatctgcttttaccgaaa ggatggctcg gccttttggt gtcttctgga catgatgccc 480 atcaaaaatgagctggggga ggtggtgctt ttcctatttt cctttaagga catctctcag 540 agtggaggcccaggacttgg ctcaccaggg atccatgggg acaataataa tcatgaaaac 600 tcccttgggaggagaggagc tagctcaaga cttaggtcca cgaggaggca gaaccggaca 660 gttctacaccggttgactgg ccactttggt cgccgggacc agggaagcgt gaaagccaat 720 agtaacgtgtttgagccaaa gccatcagtg cctgagtaca aagtggcctc cgtggggggc 780 tcccgctgcctgctcctcca ctacagcatc cccaaggctg tctgggacgg tctcatcctt 840 ctcgctacgttctacgtcgc ggtcaccgtc ccttacaacg tctgcttcgc tggtgatgac 900 gacacccccatcacgtcccg acacaccctt gtcagtgaca tcgctgtgga gatgctcttc 960 atcctggacatcatcttgaa cttccgcacc acctacgtgt cccagtcggg ccaggtggtt 1020 tctgctcctcggtccattgg cctccactac ctggccacct ggttcttcgt ggacctcatt 1080 gctgctttgccctttgacct gctgtatgtc ttcaacatca ctgtgacctc gctggtacat 1140 ctgctgaaaaccgtgcggct cctgcggttg ctgaggctgc tgcagaagct agagcggtac 1200 tctcagtgcagcgcggtggt gctcacgctg ctcatgtccg tctttgcact ccttgcccac 1260 tggatggcctgcgtctggta tgtcatcggg cgccgggaga tggaggccaa tgacccgctg 1320 ctctgggacattggttggtt gcatgagctg ggtaagcggc tggaggagcc ttatgtcaat 1380 ggctcggccggtggaccatc tcggcgcagt gcctacatcg ccgcgctgta cttcacgctg 1440 agcagcctcaccagtgtagg cttcggcaac gtttgtgcca acactgacgc tgagaagatc 1500 ttctccatctgcacgatgct cataggcgcg ctgatgcacg cggtggtgtt tgggaatgtc 1560 acagccatcatccagcgcat gtactcccga cgctcgctct accacagccg catgaaggat 1620 ctcaaggacttcatccgagt gcatcgtctg ccccgcccac tcaagcagcg catgctcgag 1680 tacttccagactacatgggc cgtcaacagc ggcatcgatg ccaacgagtt actgcgtgac 1740 ttcccggatgagctgcgagc tgacatcgcc atgcacctga atcgggagat cctgcagctg 1800 cctttgtttggagcagcaag caggggctgc cttcgtgccc tctccctgca catcaagacc 1860 tcattttgtgctcctgggga gttcctgcta cgccgtgggg atgccctgca ggcacactac 1920 tatgtctgctctggctctct tgaggtgctc cgagacaaca cggtgctggc catccttgga 1980 aagggggacttgattggggc agacatccct gagttggggc aggagcctgg ggcaggggca 2040 ggctgcgtgctgaagaccag cgctgatgtg aaagcactga cttactgcgg cctgcagcag 2100 ctgagcagccgagggctggc cgaggtcctt cggttgtatc cggaatatgt ggctgccttc 2160 agggctggcctaccccggga cctaaccttc aacctgcgcc aaggctctga aaacaatggc 2220 ctcggccgcttctcacgttc tcctcgactc tcccaggcac gctccgacac tcttggttcc 2280 tcctcagacaagactctgcc atccatcaca gaaaccgagg gtggcatgga gcctggggct 2340 ggttccaagccccgtcggcc cctcctgctg cccaacctca gtccagcacg acctcggggg 2400 tccctggtcagccttttggg cgaagagctg cccccattct cagcccttgt ctcctctcct 2460 tccctgtccccaactccttc ccctgccctg gctggccggg gttcaagtcc ctccctgcac 2520 gggccccccaggggctctgc tgcctggaag cccccccagc tcctcacccc cccactggga 2580 acatttggacctccggacct cagtccccgg atcgtggatg gcattgagga ctccagtaac 2640 acagctgaggctcctacatt ccggttcagc aagaggccgg agcccaccag aacccgttca 2700 caggctcccctttcaggccc taggctcagc cgggaactgg ccacagaggc agcagaggag 2760 gtgaaggaaaaggtctgcag gctgaaccaa gagatttcca gactcaacca ggaagtgtct 2820 cagctgagccgggagcttcg ccaagtgatg ggcctcttac aggccaggct gggtccccca 2880 agtcacccacctgactccac ttggctccca gaccttcctt gtccccatca gagaccgcca 2940 tgcatctctcctcatatgtc tggacctcca cctggtctcc agaatactac acttgctgta 3000 gtccactgtccagccagtgt tgggacagtg gagatagggg ccaccccctc agagctgagg 3060 tcttccatggtgccaccctt tccctcagaa cctgatcctc ttggaccctc tccagtgcca 3120 gaggcttctcctctgacccc aagcctcctg aagcacagct tccagtctgg gtcagacaca 3180 ttccactgaccctggccttg ggcccaggcc tgtctggggt gggcttaatt acctgccatc 3240 cagggaggagctgggctcct tggcctcttg ccttggggtc agcagctgcc agctggtctg 3300 gttggctctggattctctgg actttttaac aatgagtggc cacatctaac cccgttccat 3360 tttctaaaccatccccccca cccgtcctac tcttgtggga ggggagtcac ctgaagccgt 3420 ggaatccagcaggcatgaaa tggaccatgg tgccctgttg ggctacgcag aggagatgtc 3480 ttactttctccccacgactt ggaggctgtc atgcaaggtg gtcccttctg ctgccagcag 3540 ctcaaagcattctagcttta cccttctgca gttcccacct ccaaacatca gtcatatctg 3600 cccccctccctcccaacatg gtctcgacct caatgaggat ctgggcaact cacaaaccct 3660 ctctggtttccagctcccct ttctcactaa acaccccagg ctcacttttg gatagaaaat 3720 aaatccatatattttt 3736 11 20 DNA Artificial Sequence Description of ArtificialSequenceprobe 11 accttcctgg acaccatcgc 20 12 20 DNA Artificial SequenceDescription of Artificial Sequenceprobe 12 ccaaacacca ccgcgtgcat 20

What is claimed is:
 1. An isolated potassium channel protein, whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 2 and SEQ ID NO: 6, and which is expressed exclusively in thebrain.
 2. An isolated polynucleotide molecule which comprises anucleotide sequence encoding an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO:
 6. 3. An isolatedpolynucleotide molecule which comprises a nucleotide sequence sclectedfrom the group consisting of the 6th to 3257th nucleotide sequence ofSEQ ID NO. 1 and, the 4th to 3057th nucleotide sequence of SEQ ID NO: 5,and a nucleotide sequence which is degenerate with respect to saidnucleotide molecule.
 4. An isolated mammalian polynucleotide moleculethat hybridizes with a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:5 under stringent conditions and encodes a brain specific potassiumchannel protein, wherein a sampie is washed twice in 2×SSC containing0.1% SDS and then subjected to a third washing step, wherein said thirdwashing step is carried out in a solution selected from the groupconsisting of 0.5×SSC containing 0.1% SDS, 0.2×SSC containing 0.1% SDS,and 0.1×SSC containing 0.1% SDS.
 5. A vector which comprises thepolynucleotide molecule according to claim
 4. 6. A vector whichcomprises the polynucleotide molecule according to claim
 2. 7. A vectorwhich comprises the polynucleotide molecule according to claim
 3. 8. Ahost cell which comprises the vector according to any one of claims 5, 6and
 7. 9. A method for producing a potassium channel protein comprisingexpressing the protein in a host cell or on a host cell surfacecontaining a vector according to any one of claims 5, 6 and
 7. 10. Anisolated potassium channel protein which is produced in a host cell oron a host cell surface containing a vector according to claim 5, whereinthe protein is encoded by the vector.